Modulators of ribosomal function and identification thereof

ABSTRACT

The invention provides methods for producing high resolution crystals of ribosomes and ribosomal subunits as well as crystals produced by such methods. The invention also provides high resolution structures of ribosomal subunits either alone or in combination with protein synthesis inhibitors. The invention provides methods for identifying ribosome-related ligands and methods for designing ligands with specific ribosome-binding properties as well as ligands that may act as protein synthesis inhibitors. Thus, the methods and compositions of the invention may be used to produce ligands that are designed to specifically kill or inhibit the growth of any target organism.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending application ofU.S. application Ser. No. 09/922,251, filed Aug. 3, 2001, and claims thebenefit of (i) U.S. Provisional Application No. 60/348,731, filed Jan.14, 2002, and (ii) U.S. Provisional Application No. 60/352,024, filedJan. 25, 2002, the disclosures of each of which are incorporated byreference herein.

GOVERNMENT LICENSE RIGHTS

Certain work described herein was supported, in part, by Federal GrantNos. NIH-GM 22778 and NIH-GM54216, awarded by the National Institutes ofHealth. The Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of proteinbiosynthesis and to modulators, for example, inhibitors, of proteinbiosynthesis. More particularly, the invention relates to: methods andcompositions for elucidating the three-dimensional structure of thelarge ribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the three-dimensional structure of the largeribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the use of such structures in the design andtesting of novel protein synthesis inhibitors; and novel proteinsynthesis inhibitors.

BACKGROUND

I. Ribosomes: Structure, Function, and Composition

Ribosomes are ribonucleoproteins which are present in both prokaryotesand eukaryotes. They comprise about two-thirds RNA and one-thirdprotein. Ribosomes are the cellular organelles responsible for proteinsynthesis. During gene expression, ribosomes translate the geneticinformation encoded in a messenger RNA into protein (Garrett et al.(2000) “The Ribosome: Structure, Function, Antibiotics and CellularInteractions,” American Society for Microbiology, Washington, D.C.).

Ribosomes comprise two nonequivalent ribonucleoprotein subunits. Thelarger subunit (also known as the “large ribosomal subunit”) is abouttwice the size of the smaller subunit (also known as the “smallribosomal subunit”). The small ribosomal subunit binds messenger RNA(mRNA) and mediates the interactions between mRNA and transfer RNA(tRNA) anticodons on which the fidelity of translation depends. Thelarge ribosomal subunit catalyzes peptide bond formation—thepeptidyl-transferase reaction of protein synthesis—and includes (atleast) two different tRNA binding sites: the A-site which accommodatesthe incoming aminoacyl-tRNA, which is to contribute its amino acid tothe growing peptide chain, and the P-site which accommodates thepeptidyl-tRNA complex, i.e., the tRNA linked to all the amino acids thathave so far been added to the peptide chain. The large ribosomal subunitalso includes one or more binding sites for G-protein factors thatassist in the initiation, elongation, and termination phases of proteinsynthesis. The large and small ribosomal subunits behave independentlyduring the initiation phase of protein synthesis; however, they assembleinto complete ribosomes when elongation is about to begin.

The molecular weight of the prokaryotic ribosome is about 2.6×10⁶daltons. In prokaryotes, the small ribosomal subunit contains a 16S(Svedberg units) ribosomal RNA (rRNA) having a molecular weight of about5.0×10⁵ daltons. The large ribosomal subunit contains a 23S rRNA havinga molecular weight of about 1.0×10⁶ daltons and a 5S rRNA having amolecular weight of about 4.0×10⁵ daltons. The prokaryotic small subunitcontains about 20 different proteins and its large subunit containsabout 35 proteins. The large and small ribosomal subunits togetherconstitute a 70S ribosome in prokaryotes.

Eukaryotic ribosomes generally are bigger than their prokaryoticcounterparts. In eukaryotes, the large and small subunits together makean 80S ribosome. The small subunit of a eukaryotic ribosome includes asingle 18S rRNA, while the large subunit includes a 5S rRNA, a 5.8SrRNA, and a 28S rRNA. The 5.8S rRNA is structurally related to the 5′end of the prokaryotic 23S rRNA, and the 28S rRNA is structurallyrelated to the remainder of the prokaryotic 23S rRNA (Moore (1998) Annu.Rev. Biophys. 27: 35-58). Eukaryotic ribosomal proteins arequalitatively similar to the prokaryotic ribosomal proteins; however,the eukaryotic proteins are bigger and more numerous (Moore (1998)supra).

II. Structural Conservation of the Large Ribosomal Subunit

While the chemical composition of large ribosomal subunits vary fromspecies to species, the sequences of their components provideunambiguous evidence that they are similar in three-dimensionalstructure, function in a similar manner, and are related evolutionarily.The evolutionary implications of rRNA sequence data available arereviewed in the articles of Woese and others in part II of RibosomalRNA. Structure, Evolution, Processing and Function in ProteinBiosynthesis, (Zimmermann and Dahlberg, eds., CRC Press, Boca Raton,Fla., 1996). The article by Garret and Rodriguez-Fonseca in part IV ofthe same volume discusses the unusually high level of sequenceconservation observed in the peptidyl transferase region of the largeribosomal subunit. The ribosomes of archeal species like Haloarculamarismortui resemble those obtained from eubacterial species like E.coli in size and complexity. However, the proteins in H. marismortuiribosomes are more closely related to the ribosomal proteins found ineukaryotes (Wool et al. (1995) Biochem. Cell Biol. 73: 933-947).

III. Determination of the Structure of Ribosomes

Much of what is known about ribosome structure is derived from physicaland chemical methods that produce relatively low-resolution information.Electron microscopy (EM) has contributed to an understanding of ribosomestructure ever since the ribosome was discovered. In the 1970s, lowresolution EM revealed the shape and quaternary organization of theribosome. By the end of 1980s, the positions of the surface epitopes ofall the proteins in the E. coli small subunit, as well as many in thelarge subunit, had been mapped using immunoelectron microscopytechniques (Oakes et al. (1986), Structure, Function and Genetics ofRibosomes, (Hardesty, B. and Kramer, G., eds.) Springer-Verlag, NewYork, N.Y., pp. 47-67; Stoeffler et al. (1986), Structure, Function andGenctics of Ribosomes, (Hardesty, B. and Kramer, G., eds.)Springer-Verlag, New York, N.Y., pp. 28-46). In the last few years,advances in single-particle cryo-EM and image reconstruction have led tothree-dimensional reconstructions of the E. coli 70S ribosome and itscomplexes with tRNAs and elongation factors to resolutions of between 15Å and 25 Å (Stark et al. (1995) Structure 3: 815-821; Stark et al.(1997) Nature 3898: 403-406; Agrawal et al. (1996) Science 271:1000-1002; Stark et al. (1997) Cell 28: 19-28). Additionally,three-dimensional EM images of the ribosome have been produced atresolutions sufficiently high so that many of the proteins and nucleicacids that assist in protein synthesis can be visualized bound to theribosome. An approximate model of the RNA structure in the large subunithas been constructed to fit a 7.5 Å resolution electron microscopic mapof the 50S subunit from E. coli and available biochemical data (Muelleret al. (2000) J. Mol. Biol. 298: 35-59).

While the insights provided by EM have been useful, it has long beenrecognized that a full understanding of ribosome structure would deriveonly from X-ray crystallography. In 1979, Yonath and Wittman obtainedthe first potentially useful crystals of ribosomes and ribosomalsubunits (Yonath et al. (1980) Biochem. Internat. 1: 428-435). By themid 1980s, scientists were preparing ribosome crystals for X-raycrystallography (Maskowski et al. (1987) J. Mol. Biol. 193: 818-822).The first crystals of 50S ribosomal subunit from H. marismortui wereobtained in 1987. In 1991, improvements were reported in the resolutionof the diffraction data obtainable from the crystals of the 50Sribosomal subunit of H. marismortui (van Bohlen, K. (1991) J. Mol. Biol.222: 11).

In 1995, low resolution electron density maps for the large and smallribosomal subunits from halophilic and thermophilic sources werereported (Schlunzen et al. (1995) Biochem. Cell Biol. 73: 739-749).However, these low resolution electron density maps proved to bespurious (Ban et al. (1998) Cell 93: 1105-1115).

The first electron density map of the ribosome that showed featuresrecognizable as duplex RNA was a 9 Å resolution X-ray crystallographicmap of the large subunit from Haloarcula marismortui (Ban et al. (1998)supra). Extension of the phasing of that map to 5 Å resolution made itpossible to locate several proteins and nucleic acid sequences, thestructures of which had been determined independently (Ban et al. (1999)Nature 400: 841-847).

At about the same time, using similar crystallographic strategies, a 7.8Å resolution map was generated of the entire Thermus thermophilusribosome showing the positions of tRNA molecules bound to its A-, P-,and E-(protein exit site) sites (Cate et al. (1999) Science 285:2095-2104), and a 5.5 Å resolution map of the 30S subunit from Tthermophilus was obtained that allowed the fitting of solved proteinstructures and the interpretation of some of its RNA features (Clemons,Jr. et al. (1999) Nature 400: 833-840). Subsequently, a 4.5 Å resolutionmap of the T thermophilus 30S subunit was published, which was based inpart on phases calculated from a model corresponding to 28% of thesubunit mass that had been obtained using a 6 Å resolution experimentalmap (Tocilj et al. (1999) Proc. Natl. Acad. Sci. USA 96: 14252-14257).

IV. Location of the Peptidyl Transferase Site in the Large RibosomalSubunit

It has been known for about 35 years that the peptidyl transferaseactivity responsible for the peptide bond formation that occurs duringmessenger RNA-directed protein synthesis is intrinsic to the largeribosomal subunit (Traut et al. (1964) J. Mol. Biol. 10: 63; Rychlik(1966) Biochim. Biophys. Acta 114: 425; Monro (1967) J. Mol. Biol. 26:147-151; Maden et al. (1968) J. Mol. Biol. 35: 333-345) and it has beenunderstood for even longer that the ribosome contains proteins as wellas RNA. In certain species of bacteria, for example, the large ribosomalsubunit contains about 35 different proteins and two RNAs (Noller (1984)Ann. Rev. Biochem. 53: 119-162; Wittmann-Liebold et al. (1990) TheRibosome: Structure, Function, and Evolution, (W. E. Hill et al., eds.)American Society for Microbiology, Washington, D.C. (1990), pp.598-616). These findings posed three related questions. Which of thealmost 40 macromolecular components of the large ribosomal subunitcontribute to its peptidyl transferase site, where is that site locatedin the large subunit, and how does it work?

By 1980, the list of components that might be part of the ribosome'speptidyl transferase had been reduced to about half a dozen proteins and23S rRNA (see Cooperman (1980) Ribosomes: Structure, Function andGenetics, (G. Chambliss et al., eds.) University Park Press, Baltimore,Md. (1980), 531-554), and following the discovery of catalytic RNAs(Guerrier-Takada et al. (1983) Cell 35: 849-857; Kruger et al. (1982)Cell 31: 147-157), the hypothesis that 23S rRNA might be its soleconstituent, which had been proposed years earlier, began to gain favor.In 1984, Noller and colleagues published affinity labeling results whichshowed that U2619 and U2620 (in E. coli: U2584, U2585) are adjacent tothe CCA-end of P-site-bound tRNA (Barta et al. (1984) Proc. Nat. Acad.Sci. USA 81: 3607-3611; Vester et al. (1988) EMBO J. 7: 3577-3587).These nucleotides appear to be part of a highly conserved internal loopin the center of domain V of 23S rRNA. The hypothesis that this loop isintimately involved in the peptidyl transferase activity was supportedby the observation that mutations in that loop render cells resistant tomany inhibitors of peptidyl transferase, and evidence implicating it inthis activity has continued to mount (see, Noller (1991) Ann. Rev.Biochem. 60: 191-227; Garrett et al. (1996) Ribosomal RNA: Structure,Evolution, Processing and Function in Protein Biosynthesis, (R. A.Zimmerman and A. E. Dahlberg, eds.) CRC Press, Boca Raton, Fla. (1996),pp. 327-355).

Definitive proof that the central loop in domain V is the sole componentof the ribosome involved in the peptidyl transferase activity hasremained elusive, however. Studies have shown that it was possible toprepare particles that retained peptidyl transferase activity byincreasingly vigorous deproteinizations of large ribosomal subunits,however, it was not possible to produce active particles that werecompletely protein-free. Nevertheless, combined with earlierreconstitution results (Franceschi et al. (1990) J. Biol. Chem. 265:6676-6682), this work reduced the number of proteins that might beinvolved to just two: L2 and L3 (see, Green et al. (1997) Annu. Rev.Biochem. 66: 679-716). More recently, Watanabe and coworkers reportedsuccess in eliciting peptidyl transferase activity from in vitrosynthesized, protein-free 23S rRNA (Nitta et al. (1998) RNA 4: 257-267),however, their observations appear not to have withstood furtherscrutiny. Thus the question still remained: is the ribosome a ribozymeor is it not?

Over the years, the location of the peptidyl transferase site in theribosome has been approached almost exclusively by electron microscopy.In the mid-1980s evidence that there is a tunnel running through thelarge ribosomal subunit from the middle of its subunit interface side toits back (Milligan et al. (1986) Nature 319: 693-695; Yonath et al.(1987) Science 236: 813-816) began to accumulate, and there has beenstrong reason to believe that polypeptides pass through it as they aresynthesized (Bernabeu et al. (1982) Proc. Nat. Acad. Sci. USA 79:3111-3115; Ryabova et al. (1988) FEBS Letters 226: 255-260; Beckmann etal. (1997) Science 278: 2123-2126). More recent cryo-EM investigations(Frank et al. (1995) Nature 376: 441-444; Frank et al. (1995) Biochem.Cell Biol. 73: 757-765; Stark et al. (1995) supra) confirmed theexistence of the tunnel and demonstrated that the CCA-ends ofribosome-bound tRNAs bound to the A- and P-sites are found in thesubunit interface end of the tunnel. Consequently, the peptidyltransferase site must be located at that same position, which is at thebottom of a deep cleft in the center of the subunit interface surface ofthe large subunit, immediately below its central protuberance.

The substrates of the reaction catalyzed at the peptidyl transferasesite of the large subunit are an aminoacyl-tRNA (aa-tRNA) and apeptidyl-tRNA. The former binds in the ribosome's A-site and the latterin its P-site. The α-amino group of the aa-tRNA attacks the carbon ofthe carbonyl acylating the 3′ hydroxyl group of the peptidyl-tRNA, and atetrahedral intermediate is formed at the carbonyl carbon. Thetetrahedral intermediate resolves to yield a peptide extended by oneamino acid esterified to the A-site bound tRNA and a deacylated tRNA inthe P-site.

This reaction scheme is supported by the observations of Yarus andcolleagues who synthesized an analogue of the tetrahedral intermediateby joining an oligonucleotide having the sequence CCdA to puromycin viaa phosphoramide group (Welch et al. (1995) Biochemistry 34: 385-390).The sequence CCA, which is the 3′ terminal sequence of all tRNAs, bindsto the large subunit by itself, consistent with the biochemical datashowing that the interactions between tRNAs and the large subunitlargely depend on their CCA sequences (Moazed et al. (1991) Proc. Natl.Acad. Sci. USA 88: 3725-3728). Puromycin is an aa-tRNA analogue thatinteracts with the ribosomal A-site, and the phosphoramide group of thecompound mimics the tetrahedral carbon intermediate. This transitionstate analogue, CCdA-phosphate-puromycin (CCdA-p-Puro), binds tightly tothe ribosome, and inhibits its peptidyl transferase activity (Welch etal. (1995) supra).

V. Structure Determination of Macromolecules Using X-Ray Crystallography

In order to better describe the efforts undertaken to determine thestructure of ribosomes, a general overview of X-ray crystallography isprovided below.

Each atom in a crystal scatters X-rays in all directions, butcrystalline diffraction is observed only when a crystal is orientedrelative to the X-ray beam so that the atomic scattering interferesconstructively. The orientations that lead to diffraction may becomputed if the wavelength of the X-rays used and the symmetry anddimensions of the crystal's unit cell are known (Blundell et al. (1976)Protein Crystallography (Molecular Biology Series), Academic Press,London). The result is that if a detector is placed behind a crystalthat is being irradiated with monochromatic X-rays of an appropriatewavelength, the diffraction pattern recorded will consist of spots, eachspot representing one of the orientations that gives rise toconstructive interference.

Each spot in such a pattern, however it is recorded, is characterized by(i) an intensity (its blackness); (ii) a location, which encodes theinformation about diffraction orientation; and (iii) a phase. If all ofthose things are known about each spot in a crystal diffraction pattern,the distribution of electrons in the unit cell of the crystal may becomputed by Fourier transformation (Blundell et al. (1976) supra), andfrom that distribution or electron density map, atomic positions can bedetermined.

Unfortunately, the phase information essential for computing electrondistributions cannot be measured directly from diffraction patterns. Oneof the methods routinely used to determine the phases of macromolecules,such as proteins and nucleic acids, is called multiple isomorphousreplacement (MIR) which involves the introduction of new X-rayscatterers into the unit cell of the crystal. Typically, these additionsare heavy atoms, which make a significant contribution to thediffraction pattern. It is important that the additions be sufficientlylow in number so that their positions can be located and that they leavethe structure of the molecule or of the crystal cell unaltered, i.e.,the crystals should be isomorphous. Isomorphous replacement usually isperformed by diffusing different heavy-metal complexes into the channelsof the preformed protein crystals. Macromolecules expose side chains(such as SH groups) in these solvent channels that are able to bindheavy metals. It is also possible to replace endogenous light metals inmetalloproteins with heavier ones, e.g., zinc by mercury, or calcium bysamarium. Alternatively, the isomorphous derivative can be obtained bycovalently attaching a heavy metal to the macromolecule in solution andthen subjecting it to crystallization conditions.

Heavy metal atoms routinely used for isomorphous replacement include butare not limited to mercury, uranium, platinum, gold, lead, and selenium.Specific examples include mercury chloride, ethyl-mercury phosphate, andosmium pentamine, iridium pentaminc. Since such heavy metals containmany more electrons than the light atoms (H, N, C, O, and S) of theprotein, the heavy metals scatter x-rays more strongly. All diffractedbeams would therefore increase in intensity after heavy-metalsubstitution if all interference were positive. In fact, however, someinterference is negative; consequently, following heavy-metalsubstitution, some spots increase in intensity, others decrease, andmany show no detectable difference.

Phase differences between diffracted spots can be determined fromintensity changes following heavy-metal substitution. First, theintensity differences are used to deduce the positions of the heavyatoms in the crystal unit cell. Fourier summations of these intensitydifferences give maps, of the vectors between the heavy atoms, theso-called Patterson maps. From these vector maps, the atomic arrangementof the heavy atoms is deduced. From the positions of the heavy metals inthe unit cell, the amplitudes and phases of their contribution to thediffracted beams of protein crystals containing heavy metals iscalculated.

This knowledge then is used to find the phase of the contribution fromthe protein in the absence of the heavy-metal atoms. As both the phaseand amplitude of the heavy metals and the amplitude of the protein aloneis known, as well as the amplitude of the protein plus heavy metals(i.e., protein heavy-metal complex), one phase and three amplitudes areknown. From this, the interference of the X-rays scattered by the heavymetals and protein can be calculated to determine if the interference isconstructive or destructive. The extent of positive or negativeinterference, with knowledge of the phase of the heavy metal, give anestimate of the phase of the protein. Because two different phase anglesare determined and are equally good solutions, a second heavy-metalcomplex can be used which also gives two possible phase angles. Only oneof these will have the same value as one of the two previous phaseangles; it therefore represents the correct phase angle. In practice,more than two different heavy-metal complexes are usually made in orderto give a reasonably good estimate of the phase for all reflections.Each individual phase estimate contains experimental errors arising fromerrors in the measured amplitudes. Furthermore, for many reflections,the intensity differences are too small to measure after one particularisomorphous replacement, and others can be tried.

The amplitudes and the phases of the diffraction data from the proteincrystals are used to calculate an electron-density map of the repeatingunit of the crystal. This map then is interpreted to accommodate theresidues of the molecule of interest. That interpretation is made morecomplex by several limitations in the data. First, the map itselfcontains errors, mainly due to errors in the phase angles. In addition,the quality of the map depends on the resolution of the diffractiondata, which, in turn, depends on how well-ordered the crystals are. Thisdirectly influences the quality of the map that can be produced. Theresolution is measured in angstrom units (Å); the smaller this numberis, the higher the resolution and, therefore, the greater the amount ofdetail that can be seen.

Building the initial model is a trial-and-error process. First, one hasto decide how a polypeptide chain or nucleic acid weaves its way throughthe electron-density map. The resulting chain trace constitutes ahypothesis by which one tries to match the density of side chains to theknown sequence of the polypeptide or nucleic acid. When a reasonablechain trace has finally been obtained, an initial model is built thatfits the atoms of the molecule into the electron density. Computergraphics are used both for chain tracing and for model building topresent the data and manipulated the models.

The initial model will contain some errors. Provided the crystalsdiffract to high enough resolution (e.g., better than 3.5 Å), most orsubstantially all of the errors can be removed by crystallographicrefinement of the model using computer algorithms. In this process, themodel is changed to minimize the difference between the experimentallyobserved diffraction amplitudes and those calculated for a hypotheticalcrystal containing the model (instead of the real molecule). Thisdifference is expressed as an R factor (residual disagreement) which is0.0 for exact agreement and about 0.59 for total disagreement.

In general, the R factor for a well-determined macromolecular structurepreferably lies between 0.15 and 0.35 (such as less than about0.24-0.28). The residual difference is a consequence of errors andimperfections in the data. These derive from various sources, includingslight variations in the conformation of the protein molecules, as wellas inaccurate corrections both for the presence of solvent and fordifferences in the orientation of the microcrystals from which thecrystal is built. This means that the final model represents an averageof molecules that are slightly different both in conformation andorientation.

In refined structures at high resolution, there are usually no majorerrors in the orientation of individual residues, and the estimatederrors in atomic positions are usually around 0.1-0.2 Å, provided thesequence of the protein or nucleic acid is known. Hydrogen bonds, bothwithin the molecule of interest and to bound ligands, can be identifiedwith a high degree of confidence.

Typically, X-ray structures can be determined provided the resolution isbetter than 3.5 Å. Electron-density maps are interpreted by fitting theknown amino acid and/or nucleic acid sequences into regions of electrondensity.

VI. The Need for Higher Resolution for the 50S Ribosomal Subunit

Although the art provides crystals of the 50S ribosomal subunit, and 9 Åand 5 Å resolution X-ray crystallographic maps of the structure of the50S ribosome, the prior art crystals and X-ray diffraction data are notsufficient to establish the three-dimensional structures of all 31proteins and 3,043 nucleotides of the 50S ribosomal subunit. Thus, theprior art crystals and maps are inadequate for the structure-baseddesign of active agents, such as herbicides, drugs, insecticides, andanimal poisons.

More detailed, higher resolution X-ray crystallographic maps arenecessary in order to determine the location and three-dimensionalstructure of the proteins and nucleotides in ribosomes and ribosomalsubunits, particularly for the 50S ribosomal subunit. An accuratemolecular structure of the 50S ribosomal subunit will not only enablefurther investigation and understanding of the mechanism of proteinsynthesis, but also the development of effective therapeutic agents anddrugs that modulate (e.g., induce or inhibit) protein synthesis.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the determination of ahigh resolution atomic structure of a ribosomal subunit, moreparticularly, a large subunit of a ribosome. In addition, the inventionis based, in part, upon the determination of high resolution atomicstructures of certain protein synthesis inhibitors, namely antibiotics,when they are interacting with the large subunit of the ribosome.

The invention provides the structure of the large subunit of a ribosomeisolated from the organism, Haloarcula marismortui. However, in view ofthe high level of sequence and structural homology between ribosomes oforganisms in different kingdoms, the structural information disclosedherein can be used to produce, using routine techniques, high resolutionstructural models of large ribosomal units for any organism of interest.

Although there is significant homology between ribosomes of differentorganisms, for example, between ribosomes of humans and certain humanpathogens, there still are differences that can be exploitedtherapeutically. For example, many clinically and commerciallysignificant protein synthesis inhibitors, for example, antibiotics suchas streptomycin, tetracycline, chloramphenicol and erythromycin,selectively target bacterial ribosomes and disrupt bacterial proteinsynthesis but at the same time do not target or otherwise significantlyaffect human ribosome function. As a result, over the years antibioticshave proven to be invaluable in the treatment of microbial infections inhumans. However, there is still an ongoing need for new proteinsynthesis inhibitors, particularly because of the development of strainsof pathogens that are resistant to known antibiotics. The informationprovided herein provides insights into the design of new proteinsynthesis inhibitors.

The invention provides computer systems containing atomic co-ordinatesthat define at least a portion of the three-dimensional structure of aribosome, more specifically, a large ribosomal subunit. In addition, theinvention provides methods of using the atomic co-ordinates to identifynew molecules that can selectively bind ribosomes, and that preferablyact as selective inhibitors of protein synthesis. In addition, theinvention provides computer systems containing atomic co-ordinates thatdefine at least a portion of certain antibiotics when interacting withtheir binding sites in the large ribosomal subunit. In addition, theinvention provides methods of using the atomic co-ordinates of theantibiotics to identify new molecules that selectively bind ribosomes,and that preferably act as selective inhibitors of protein synthesis. Inaddition, the invention provides new families of protein synthesisinhibitors. Each of these aspects of the invention is discussed in moredetail below.

In one aspect, the invention provides a computer system comprising: (a)a memory having stored therein data indicative of atomic co-ordinatesderived from an electron density map having a resolution of at leastabout 4.5 Å, more preferably of at least about 3.0 Å, and mostpreferably of about 2.4 Å and defining a ribofunctional locus of a largesubunit of a ribosome; and (b) a processor in electrical communicationwith the memory, the processor comprising a program for generating athree-dimensional model representative of the ribofunctional locus. In apreferred embodiment, the computer system further comprises a device,for example, a computer monitor, or terminal for providing a visualrepresentation of the molecular model. In another preferred embodiment,the processor further comprises one or more programs to facilitaterational drug design.

In a preferred embodiment, the computer system further comprises atleast a portion of the atomic co-ordinates deposited at the Protein DataBank under accession number PDB ID: 1FFK (large ribosomal subunit), 1FFZ(large ribosomal subunit complexed with CCdA-p-Puro), 1FG0 (largeribosomal subunit complexed with a mini-helix analogue ofaminoacyl-tRNA), 1JJ2 (large ribosomal structure), 1K73 (large ribosomalsubunit complexed with anisomycin), 1KC8 (large ribosomal subunitcomplexed with blasticidin), 1K8A (large ribosomal subunit complexedwith carbomycin), 1KD1 (large ribosomal subunit complexed withspiramycin); or 1K9M (large ribosomal subunit complexed with tylosin) orrecorded on compact disk, Disk No. 1 of 1.

In a preferred embodiment, the atomic co-ordinates further define atleast a portion of a protein synthesis inhibitor, for example, anantibiotic, more specifically an antibiotic selected from the groupconsisting of anisomycin, blasticidin, carbomycin A, sparsomycin,spiramycin, tylosin, virginiamycin M, azitbromycin, linezolid,chloramphenicol and erythromycin, complexed with a ribofunctional locus.More specifically, the invention provides atomic co-ordinates of thelarge ribosomal subunit together the atomic co-ordinates of antibioticsinteracting with the large ribosomal subunit. These atomic co-ordinatesare recorded on compact disk, Disk No. 1, and correspond to: largeribosomal subunit complexed with anisomycin (file name: amsomysin.pdb orANISOMYC.PDB); large ribosomal subunit complexed with blasticidin (filename: blasticidin.pdb or BLASTICI.PDB); large ribosomal subunitcomplexed with carbomycin (file name: carbomycin.pdb or CARBOMYC.PDB);large ribosomal subunit complexed with tylosin (file name: tylosin.pdbor TYLOSIN.PDB); large ribosomal subunit complexed with sparsomycin(file name: sparsomycin.pdb or SPARSOMY.PDB); large ribosomal subunitcomplexed with virginiamycin M (file name: virginiamycin.pdb orVIRGLNIA.PDB); large ribosomal subunit complexed with spiramycin (filename: spiramycin.pdb or SPIRAMYC.PDB); large ribosomal subunit complexedwith azithromycin (file name: AZITHROM.PDB or azithromycin.pdb); orlarge ribosomal subunit complexed with linezolid (file name:LTNEZOLI.PDB or linezolid.pdb); or large ribosomal subunit complexedwith erythromycin (file name: erythromycin.pdb).

In a preferred embodiment, the ribofunctional locus comprises at least aportion of an active site in the ribosomal subunit, for example, atleast a portion of one or more of: a peptidyl transferase site (aportion of which may be defined by a plurality of residues set forth inTable 5A or 5B); an A-site (a portion of which may be defined by aplurality of residues set forth in Table 6A or 6B); a P-site (a portionof which may be defined by a plurality of residues set forth in Table 7Aor 7B); a polypeptide exit tunnel (a portion of which may be defined bya plurality of residues set forth in Table 8A or 8B, Table 9, or Table10); or an antibiotic binding domain (a portion of which may be definedby a plurality of residues set forth in Table 11A or 11B, Table 12A or12B, Table 13A or 13B, Table 14A or 14B, Table 15A or 15B, Table 16A or16B, Table 17A or 17B, Table 18A or 18B, Table 19A or 19B, or Table 20Aor 20B). A plurality of residues shall be considered to include at least3 residues, preferably at least 5 residues, and more preferably at least10 residues. The ribofunctional locus may be defined by atoms ofribosomal RNA, one or more ribosomal proteins, or a combination ofribosomal RNA and one or more ribosomal proteins.

In another preferred embodiment, the atomic co-ordinates are produced bymolecular modeling. Using the atomic co-ordinates provided herein, theskilled artisan may generate models of any ribosome of interest usingconventional techniques, for example, conventional homology modeling,and or molecular replacement techniques. In another embodiment, theatomic co-ordinates are produced by homology modeling using at least aportion of the atomic co-ordinates deposited at the Protein Data Bankunder accession number PDB ID: 1FFK, 1FFZ, 1FG0, 1JJ2, 1K73, 1KC8, 1K8A,1KD1, or 1K9M, or any of the atomic co-ordinates included in compactdisk, Disk No. 1. In another embodiment, the atomic co-ordinates areproduced by molecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, 1JJ2, 1K73, 1KC8, 1K8A, 1KD1 or 1K9M, or anyof the atomic co-ordinates recorded on compact disk, Disk No. 1.

In a preferred embodiment, the atomic co-ordinates define residues thatare conserved between ribosomes or ribosomal subunits of pathogens, forexample, prokaryotic organisms, and, optionally but more preferably, arealso absent from ribosomes or ribosomal subunits of a host organism, forexample, a human. In another preferred embodiment, the atomicco-ordinates may define residues that are conserved between ribosomes orribosomal subunits of prokaryotic organisms, for example, bacteria, and,optionally but more preferably, are also absent from ribosomal subunitsof eukaryotes, for example, a mammal, more preferably, a human. Thisinformation can be used, for example, via the use of one or moremolecular models, to identify targets for rational drug design that maybe exploited to develop new molecules, for example, protein synthesisinhibitors, that disrupt protein synthesis in a pathogen, for example, abacteria, but do not disrupt or otherwise substantially affect proteinsynthesis in a host organism, for example, a human.

In another aspect, the invention provides a variety of methods fordesigning, testing and refining new molecules via rational drug design.For example, the invention provides a method that comprises the stepsof: (a) providing a model, for example, a molecular model, having aribofunctional locus of a large subunit of a ribosome, wherein the modelis defined by the spatial arrangement of atoms derived from an electrondensity map having a resolution of at least about 4.5 Å, more preferablyto at least about 3.0 Å, and most preferably to about 2.4 Å; and (b)using the model to identify a candidate molecule having a surfacecomplementary to the ribofunctional locus. Preferably, the candidatemolecule stereochemically interfits and more preferably binds with theribofunctional locus of the large subunit of the ribosome.

In a preferred embodiment, the method comprises one or more additionalsteps of: producing the candidate molecule identified in such a method;determining whether the candidate molecule, when produced, modulates(for example, induces or reduces) ribosomal activity; identifying amodified molecule; producing the modified molecule; determining whetherthe modified molecule, when produced, modulates ribosomal activity; andproducing the modified molecule for use either alone or in combinationwith a pharmaceutically acceptable carrier. The candidate moleculeand/or the modified molecule may be an antibiotic or antibioticanalogue, for example, a macrolide antibiotic or a macrolide analogue.

In a preferred embodiment, the ribofunctional locus used in such amethod comprises at least a portion of an active site in the ribosomalsubunit. In another preferred embodiment, the ribofunctional locus isdefined by at least a portion of one or more of: a peptidyl transferasesite (a portion of which may be defined by a plurality of residues setforth in Table 5A or Table 5B); an A-site (a portion of which may bedefined by a plurality of residues set forth in Table 6A or Table 6B); aP-site (a portion of which may be defined by a plurality of residues setforth in Table 7A or Table 7B); a polypeptide exit tunnel (a portion ofwhich may be defined by a plurality of residues set forth in Table 8A,Table 8B, Table 9, Table 10); or an antibiotic binding domain (a portionof which may be defined by a plurality of residues set forth in Table11A, Table 11B, Table 12A, Table 12B, Table 13A, Table 13B, Table 14A,Table 14B, Table 15A, Table 15B, Table 16A, Table 16B, Table 17A, Table17B, Table 18A, Table 18B, Table 19A, Table 19B, Table 20A or Table20B). The ribofunctional locus may be defined by atoms of ribosomal RNA,one or more ribosomal proteins, or a combination of ribosomal RNA andone or more ribosomal proteins.

In another preferred embodiment, the atomic co-ordinates are used toproduce a molecular model in an electronic form. The atomic co-ordinatespreferably are produced by molecular modeling. In another embodiment,the atomic co-ordinates are produced by homology modeling using at leasta portion of the atomic co-ordinates deposited at the Protein Data Bankunder accession number PDB ID: 1FFK, 1FFZ, 1FG0, 1JJ2, 1K73, 1KC8, 1K8A,1KD, or 1K9M, or the atomic co-ordinates recorded on compact disk, DiskNo. 1. In another embodiment, the atomic co-ordinates are produced bymolecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, 1JJ2, 1K73, 1KC8, 1K8A, 1KD1, or 1K9M or anyof the atomic co-ordinates included on compact disk, Disk No. 1.

In a preferred embodiment, the atomic co-ordinates may define residuesthat are conserved among ribosomes or ribosomal subunits of pathogens,for example, prokaryotic organisms, and, optionally but more preferably,are also absent in ribosomes or ribosomal subunits of a host organism,for example, a human. In another preferred embodiment, the atomicco-ordinates may define residues that are conserved between ribosomes orribosomal subunits of prokaryotic organisms, for example, bacteria, and,optionally but more preferably, are also absent from ribosomes orribosomal subunits of eukaryotes, for example, a mammal, more preferablya human. This information can be used, for example, via the use of oneor more molecular models, to identify targets for rational drug designthat may be exploited to develop new molecules, for example, proteinsynthesis inhibitors, that disrupt protein synthesis in a pathogen, forexample, a bacteria but do not disrupt or otherwise substantially affectprotein synthesis in a host organism, for example, a human.

In a preferred embodiment, the computer system further comprises atleast a portion of the atomic co-ordinates deposited at the Protein DataBank under accession number PDB ID: 1FFK (large ribosomal subunit), 1FFZ(large ribosomal subunit complexed with CCdA-p-Puro), 1FG0 (largeribosomal subunit complexed with a mini-helix analogue ofaminoacyl-tRNA), 1JJ2 (large ribosomal subunit), 1K73 (large ribosomalsubunit complexed with anisomycin), 1KC8 (large ribosomal subunitcomplexed with blasticidin), 1K8A (large ribosomal subunit complexedwith carbomycin), 1KD1 (large ribosomal subunit complexed withspiramycin); or 1K9M (large ribosomal subunit complexed with tylosin),or recorded on compact disk, Disk No. 1.

In another aspect, the invention provides a computer system comprising:(a) a memory having stored therein data indicative of atomicco-ordinates derived from an electron density map defining at least aportion of a protein synthesis inhibitor when the protein synthesisinhibitor is interacting with a ribofunctional locus of a large subunitof a ribosome; and (b) a processor in electrical communication with thememory, the processor comprising a program for generating athree-dimensional model representative of at least a portion of theprotein synthesis inhibitor. In a preferred embodiment, the computersystem further comprises a device, for example, a computer monitor, orterminal for providing a visual representation of the molecular model.In another preferred embodiment, the processor further comprises one ormore programs to facilitate rational drug design.

In a preferred embodiment, the atomic co-ordinates further define atleast a portion of a protein synthesis inhibitor, for example, anantibiotic, more specifically an antibiotic selected from the groupconsisting of anisomycin, blasticidin, carbomycin A, sparsomycin,spiramycin, tylosin, virginiamycin M, azithromycin, linezolid anderythromycin, complexed with a ribofunctional locus. In a preferredembodiment, the ribofunctional locus comprises at least a portion of anactive site of a ribosome, for example, at least a portion of one ormore of (i) a peptidyl transferase site, (ii) an A-site, (iii) a P-site,(iv) a polypeptide exit tunnel.

More specifically, the invention provides atomic co-ordinates ofantibiotics interacting with the large ribosomal subunit. These atomicco-ordinates are recorded on compact disk, Disk No. 1, and correspondto: large ribosomal subunit complexed with anisomycin (file name:anisomysin.pdb or ANISOMYC.PDB); large ribosomal subunit complexed withblasticidin (file name: blasticidin.pdb or BLASTICI.PDB); largeribosomal subunit complexed with carbomycin A (file name: carbomycin.pdbor CARBOMYC.PDB); large ribosomal subunit complexed with tylosin (filename: tylosin.pdb or TYLOSIN.PDB); large ribosomal subunit complexedwith sparsomycin (file name: sparsomycin.pdb or SPARSOMY.PDB); largeribosomal subunit complexed with virginiamycin M (file name:virginiamycin.pdb or VIRGLNIA.PDB); large ribosomal subunit complexedwith spiramycin (file name: spiramycin.pdb or SPIRAMYC.PDB); largeribosomal subunit complexed with azithromycin (file name: AZITHROM.PDBor azithromycin.pdb); or large ribosomal subunit complexed withlinezolid (file name: LINEZOLI.PDB or linezolid.pdb); or large ribosomalsubunit complexed with erythromycin (file name: erythromycin.pdb).

In another aspect, the invention provides a method of identifying a leadcandidate for a new protein synthesis inhibitor. The method comprisesthe steps of (a) providing a molecular model of at least a portion of aprotein synthesis inhibitor when the protein synthesis inhibitor isinteracting with a ribofunctional locus of a large subunit of aribosome; and (b) using the model to identify the lead candidate. In apreferred embodiment, the lead candidate is capable of interacting, andstill more preferably binding a ribofunctional locus.

In another preferred embodiment, the method comprises the additionalstep of producing the lead compound. After synthesis, the lead compoundcan be tested for biological activity, for example, modulation ofribosome activity in an in vitro assay or growth inhibition ofmicro-organisms of interest. Based on the results of such studies, it ispossible to determine structure-activity relationships, which may thenbe used to design further modifications of the lead compound in order toimprove a particular feature of interest. The modified lead compoundsthen can be produced and assessed for biological activity, as before.Once a compound of interest has been designed, synthesized and testedfor activity, it may then be produced in commercially feasiblequantities for use as a pharmaceutical.

In a preferred embodiment, the starting molecule (i.e., the proteinsynthesis inhibitor), the lead compound and the penultimate compound isan antibiotic or antibiotic analogue. In another embodiment, the leadcompound and also the penultimate compound is a hybrid antibiotic (i.e.,comprises a portion of a first antibiotic and a portion of a second,different antibiotic).

In another preferred embodiment, the model used in the practice of theinvention is a model of an antibiotic selected from the group consistingof anisomycin, blasticidin, carbomycin A, sparsomycin, spiramycin,tylosin, virginiamycin M, azithromycin, linezolid, or erythromycin. Themodel preferably comprises a portion of the atomic co-ordinates recordedon compact disk, Disk No. 1 of 1 under file name: anisomycin.pdb,blasticidin.pdb, carbomycin.pdb, sparsomycin.pdb, spiramycin.pdb,tylosin.pdb, virginiamycin.pdb, ANISOMYC.PDB, BLASTICI.PDB,CARBOMYC.PDB, SPARSOMY.PDB, SPIRAMYC.PDB, TYLOSIN.PDB, VIRGLNIA.PDB,AZITHROM.PDB, LINEZOLI.PDB, azithromycin.pdb, linezolid.pdb, orerythromycin.pdb. In another preferred embodiment, the penultimatecompound is an analogue of an antibiotic selected from the groupconsisting of anisomycin, blasticidin, carbomycin A, sparsomycin,spiramycin, tylosin, virginiamycin M, azithromycin, linezolid, orerythromycin.

In a preferred embodiment, the ribofunctional locus comprises at least aportion of an active site of a ribosome, for example, at least a portionof one or more of (i) a peptidyl transferase site, (ii) an A-site, (iii)a P-site, and (iv) a polypeptide exit tunnel. In another embodiment, themolecular model useful in the practice of the invention is in anelectronic form, in which case the molecular model preferably isgenerated by molecular modeling.

In another aspect, the invention provides new protein synthesisinhibitors that disrupt the function of a target ribosome. Theseinhibitors can be readily designed and tested as disclosed herein.

One type of protein synthesis inhibitor of the invention comprises: afirst binding domain having a surface, for example, a solvent accessiblesurface, that mimics or duplicates a surface of a known first molecule,for example, a first antibiotic, that binds with a first contact site,for example, a first ribofunctional locus, in or on a large ribosomalsubunit; and a second binding domain having a surface, for example, asolvent accessible surface, that mimics or duplicates a surface of aknown second molecule, for example, a second antibiotic, that binds witha second contact site, for example, a second ribofunctional locus, in oron the ribosomal subunit. The first domain is attached to the seconddomain so as to permit both the first domain and the second domain tobind simultaneously with their respective contact sites within or on theribosomal subunit so as to disrupt protein synthesis in a ribosomalsubunit. In a preferred embodiment, the protein synthesis inhibitor hasa molecular weight of less than about 1,500 and an IC₅₀ of lower thanabout 50 μM.

Another type of protein synthesis inhibitor is a synthetic, engineeredmolecule that comprises: (i) a binding domain having a surface, forexample, a solvent accessible surface, that mimics or duplicates asolvent accessible surface of a known molecule, for example, a firstknown antibiotic, which binds with a contact site, for example, aribofunctional locus in or on a ribosomal subunit; and (ii) a noveleffector domain attached to the binding domain which, upon binding ofthe binding domain with the contact site, occupies a space within oradjacent to the ribosomal subunit thereby disrupting protein synthesisin the ribosomal subunit. In a preferred embodiment, the proteinsynthesis inhibitor has a molecular weight of less than about 1,500 andan IC₅₀ of lower than about 50 μM.

In another aspect, the invention provides new protein synthesisinhibitors, for example, a molecule capable of contacting at least threeresidues but less than thirteen contact residues in Table 11A thattogether define an anisomycin binding pocket of a large ribosomalsubunit, a molecule capable of contacting at least three residues butless than ten contact residues in Table 12A that together define ablasticidin binding pocket of a large ribosomal subunit, a moleculecapable of contacting at least three residues but less than sixteencontact residues in Table 13A that together define a carbomycin Abinding pocket of a large ribosomal subunit, a molecule capable ofcontacting at least three residues but less than twenty contact residuesin Table 14A that together define a tylosin binding pocket of a largeribosomal subunit, a molecule capable of contacting at least threeresidues but less than nine contact residues in Table 15A that togetherdefine a sparsomycin binding pocket of a large ribosomal subunit, amolecule capable of contacting at least three residues but less thanthirteen contact residues in Table 16A that together define avirginiamycin M binding pocket of a large ribosomal subunit, a moleculecapable of contacting at least three residues but less than fifteencontact residues in Table 17A that together define a spiramycin bindingpocket of a large ribosomal subunit, a molecule capable of contacting atleast three residues but less than thirteen contact residues in Table18A that together define an erythromycin binding pocket of a largeribosomal subunit, a molecule capable of contacting at least three butless than eleven contact residues in Table 19A that together define anazithromycin binding pocket of a large ribosomal subunit, or a moleculecapable of contacting at least three residues but less than fifteencontact residues in Table 20A that together define a linezolid bindingpocket of a large ribosomal subunit. The contact residues are thoseresidues in the large ribosomal subunit that are in van der Waalscontact with the antibiotic of interest.

In yet another aspect, the invention provides a protein synthesisinhibitor comprising, for example, a molecule capable of contacting aplurality of residues in Table 11A that together define an anisomycinbinding pocket of a large ribosomal subunit, but lacking one or moreatoms present in the anisomycin molecule, the atomic co-ordinates ofwhich are recorded on Disk No. 1 under file name ANISOMYC.PDB; amolecule capable of contacting a plurality of residues in Table 12A thattogether define a blasticidin binding pocket of a large ribosomalsubunit, but lacking one or more atoms present in the blasticidinmolecule, the atomic co-ordinates of which are recorded on Disk No. 1under file name BLASTICI.PDB; a molecule capable of contacting aplurality of residues in Table 13A that together define a carbomycin Abinding pocket of a large ribosomal subunit, but lacking one or moreatoms present in carbomycin A, the atomic co-ordinates of which arerecorded on Disk No. 1 under file name CARBOMYC.PDB; a molecule capableof contacting a plurality of residues in Table 14A that together definea tylosin binding pocket of a large ribosomal subunit, but lacking oneor more atoms present in tylosin, the atomic co-ordinates of which arerecorded on Disk No. 1 under file name TYLOSIN.PDB; a molecule capableof contacting a plurality of residues in Table 15A that together definea sparsomycin binding pocket of a large ribosomal subunit, but lackingone or more atoms present in sparsomycin, the atomic co-ordinates ofwhich are recorded on Disk No. 1 under file name SPARSOMY.PDB; amolecule capable of contacting a plurality of residues in Table 16A thattogether define a virginiamycin M binding pocket of a large ribosomalsubunit, but lacking one or more atoms present in virginiamycin M, theatomic co-ordinates of which are recorded on Disk No. 1 under file nameVIRGLNIA.PDB; a molecule capable of contacting a plurality of residuesin Table 17A that together define a spiramycin binding pocket of a largeribosomal subunit, but lacking one or more atoms present in spiramycin,the atomic co-ordinates of which are recorded on Disk No. 1 under filename SPIRAMYC.PDB; a molecule capable of contacting a plurality ofresidues in Table 18A that together define an erythromycin bindingpocket of a large ribosomal subunit, but lacking one or more atomspresent in erythromycin, the atomic co-ordinates of which are recordedon Disk No. 1 under file name erythromycin.pdb; a molecule capable ofcontacting a plurality of residues in Table 19A that together define anazithromycin binding pocket of a large ribosomal subunit, but lackingone or more atoms present in azithromycin, the atomic co-ordinates ofwhich are recorded on Disk No. 1 under file name azithromycin.pdb; or amolecule capable of contacting a plurality of residues in Table 20A thattogether define a linezolid binding pocket of a large ribosomal subunit,but lacking one or more atoms present in linezolid, the atomicco-ordinates of which are recorded on Disk No. 1 under file namelinezolid.pdb.

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following figures, detailed descriptionand claims. Further advantages are evident from the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Color renditions similar to some of the following figures can be found,for example, in Ban et al. (2000) Science 289: 905-920; or Nissen et al.(2000) Science 289: 920-930.

The objects and features of the invention may be more fully understoodby reference to the drawings described below:

FIGS. 1(A)-(E) show the electron density from a 2.4 Å resolutionelectron density map. Specifically, FIG. 1(A) shows a stereo view of ajunction between 23S rRNA domains II, III, and IV. FIG. 1(B) shows theextended region of protein L2 interacting with surrounding RNA. FIG.1(C) shows in detail the L2 region with a bound Mg²⁺ ion. FIG. 1(D)shows in detail L2 with amino acid side chains. FIG. 1(E) shows helices94-97 from domain 6.

FIG. 2 shows the H. marismortui large ribosomal subunit in the crownview. The subunit is shown in the crown view, with its L7/L12 stalk tothe right, its L1 stalk to the left, and its central protuberance (CP)up. In this view, the surface of the subunit that interacts with thesmall ribosomal subunit faces the reader. RNA is shown in gray in aspace-filling rendering. The backbones of the proteins visible arerendered in gold. A transition state analogue bound to the peptidyltransferase site of the subunit is indicated in green. The particle isapproximately 250 Å across.

FIGS. 3(A)-(B) show the secondary structure of the 23S rRNA from H.marismortui. The secondary structure of this 23S rRNA is shown in astandardized format. FIG. 3(A) shows the 5′ half of the large subunitrRNA. FIG. 3(B) show the 3′ half of the large subunit rRNA. This diagramshows all the base pairings seen in the crystal structure of the largesubunit that are stabilized by at least two hydrogen bonds. Pairingsshown in red were predicted and are observed. Those shown in green werepredicted, but are not observed. Interactions shown in blue areobserved, but were not predicted. Bases shown in black do not appear tobe involved in pairing interactions. Sequences that cannot be visualizedin the 2.4 Å resolution electron density map are depicted in gray withthe secondary structures predicted for them.

FIGS. 4(A)-(L) show the tertiary structures of the RNA domains in the Hmarismortui large ribosomal subunit, its RNA as a whole, and schematicsof its RNAs. Specifically, FIGS. 4(A) and 4(B) show the RNA structure ofthe entire subunit. Domains are color coded as shown in the schematic ofFIG. 5(C). FIG. 4(A) shows the particle in the crown view. FIG. 4(B)shows the image in FIG. 4(A) rotated 180° about an axis runningvertically in the plane of the image. FIGS. 4(C) and 4(D) show aschematic diagram of 23S rRNA and the secondary structure of 5S rRNA.FIG. 4(C) shows a schematic diagram of 23S rRNA secondary structure ofFIG. 3 with helices numbered according to Leffers et al. ((1987) J. Mol.Biol. 195: 43-61), and the domains of the molecule are indicated bycolor shading. FIG. 4(D) shows the secondary structure of 5S rRNA from Hmarismortui. Thick lines joining bases represent Watson-Crick pairing.Bases joined by a lower case “o” indicate non-Watson-Crick pairing.Bases joined by thin lines interact via a single hydrogen bond. Basesshown in black are not paired. Bases shown in red are phylogeneticallypredicted pairing that have now been confirmed (Symanski et al. (1998)Nucl. Acids Res. 26: 156-159). Pairs shown in blue are observed, butwere not predicted, and pairs shown in green were predicted but are notobserved. FIGS. 4(E) through 4(L) show stereo views of the RNA domainsin the 23S rRNA and of 5S rRNA. Each domain is color-coded from its 5′end to its 3′ end to facilitate the viewer following its trajectory inthree-dimensions. The surfaces where the most important inter-domaininteractions occur are shown in mono to the right of the stereo views.FIG. 4(E) shows domain I; FIG. 4(F) shows domain II; FIG. 4(G) showsdomain III; FIG. 4(H) shows domain IV; FIG. 4(I) shows domain V, crownview; FIG. 4(J) shows domain V, back view; FIG. 4(K) shows domain VI;and FIG. 4(L) shows 5S rRNA.

FIGS. 5(A)-(C) show conservations and expansions in the 23S rRNA of H.marismortui. The generality of the RNA in these images is gray.Sequences that are found to be >95% conserved across the threephylogenetic kingdoms are shown in red. Sequences where expansion in thebasic 23S structure is permitted are shown in green (Gutell et al.(2000) supra). Specifically, FIG. 5(A) shows the particle rotated withrespect to the crown view so that its active site cleft can be seen.FIG. 5(B) shows the crown view. FIG. 5(C) shows the back view of theparticle, i.e., the crown view rotated 180° about its vertical axis.

FIGS. 6(A)-(I) show structures of some large subunit ribosomal proteinsthat have non-globular extensions. Only the backbones of the proteinsare shown. The globular domains of these proteins are shown in green,and their non-globular extensions are depicted in red. The positions ofthe zinc ions in L44e and L37e are also indicated. FIG. 6(A) shows L2;FIG. 6(B) shows L3; FIG. 6(C) shows L39; FIG. 6(D) shows L4; FIG. 6(E)shows L15; FIG. 6(F) shows L21e; FIG. 6(G) shows L44e; FIG. 6(H) showsL37e; FIG. 6(I) shows L19;

FIGS. 7(A)-(C) show proteins that appear on the surface of the largeribosomal subunit. The RNA of the subunit is shown in gray, as in FIG.2, and protein backbones are shown in gold. Specifically, FIG. 7(A)shows the subunit in the crown view of the subunit. FIG. 7(B) shows theback side of the subunit in the crown view orientation. FIG. 7(C) showsthe bottom view; the end of the peptide tunnel appears in the center ofthis image. The proteins visible in each image are identified in thesmall images at the lower left corner of the Figure.

FIGS. 8(A)-(F) show the protein distribution and protein-RNAinteractions in the large ribosomal subunit. Specifically, FIG. 8(A)shows the structures of proteins in the neighborhood of the end of thepeptide tunnel and how they relate to the RNA sequences with which theyinteract. Protein L22 extends a long β hairpin extension inside the 23SrRNA. L24 has a similar extension but the entire protein is on thesurface of the particle. L39 is the only protein in the subunit thatlacks tertiary structure, while L37e has both NH₂ and COOH terminalextensions. L19 is unique in having two globular domains on the surfaceof the subunit connected by an extended sequence that weaves through theRNA. The end of L39 (green) actually enters the tunnel, while L37e (red)is entirely surrounded by RNA. FIG. 8(B) shows the non-globularextensions of L2 and L3 reaching through the mass of 23S rRNA towardsthe peptidyl transferase site, which is marked by a CCdA-p-puromycinmolecule. FIG. 8(C) shows L22 interacting with portions of all six ofthe domains of 23S rRNA. FIG. 8(D) shows a schematic of 23S rRNA showingthe locations of the sequences that make at least van der Waals contactwith protein (red). FIG. 8(E) shows a stereo view of the proteins of thelarge ribosomal subunit with all the RNA stripped away. Proteins arecolor red as an aid to visualization only. FIG. 8(F) shows a crosssection of the subunit in the area of the tunnel exit. Protein L22 isshown as ribbons in red, and the β hairpin loop where mutations confererythromycin resistance is shown in orange. Atoms on the surface areshown in gray, protein atoms are shown in green, and atoms at the sliceinterface are shown in blue.

FIGS. 9(A)-(C) show chemical structures of ribosome peptidyl transferasesubstrates and analogues. Specifically, FIG. 9(A) shows the tetrahedralcarbon intermediate produced during peptide bond formation; thetetrahedral carbon is indicated by an arrow. FIG. 9(B) shows thetransition state analogue formed by coupling the 3′ OH of CCdA to theamino group of the O-methyl tyrosine residue of puromycin via aphosphate group, CCdA-p-Puro (Welch et al. (1995) supra). FIG. 9(C)shows an amino-N-acylated mini helix constructed to target the A-site.The oligonucleotide sequence 5′ phosphateCCGGCGGGCUGGUUCAAACCGGCCCGCCGGACC 3′ (SEQ ID NO: 1) puromycin shouldform 12 base pairs. The construct was based on a mini helix which is asuitable substrate for amino-acylation by Tyr-tRNA synthetase. The 3′ OHof its terminal C is coupled to the 5′ OH of the N6-dimethyl A moiety ofpuromycin by a phosphodiester bond.

FIGS. 10(A)-(C) show experimentally phased electron density maps of thesubstrate analogue complexes at 3.2 Å resolution, with modelssuperimposed (oxygen, red; phosphorus, purple; nitrogen, blue; andcarbon, green for rRNA and yellow for substrate). Specifically, FIG.10(A) shows an F_(o)(complex)-F_(o)(parent) difference electron densitymap with a skeletal model of CCdA-p-Puro superimposed. FIG. 10(B) showsa 2F_(o)(complex)-F_(o)(parent) electron density map of the CCdA-p-Puroin the active site region with the structures of the ribosome andinhibitor superimposed showing the proximity of the N3 of A2486 (2451)to the phosphate, non-bridging oxygen in this complex. FIG. 10(C) showsan F_(o)(complex)-F_(o)(parent) differences electron density map of thetRNA acceptor stem analogue with a skeletal model of CCpurosuperimposed. There is density only for the ribose and phosphate of C74and none for the rest of the RNA hairpin.

FIGS. 11(A) and (B) show a combined model of the CCA portion of the minihelix bound to the A-site and CCdA-p-Puro bound to the A- and P-sites,color coded as in FIG. 2. Specifically, FIG. 11(A) shows thebase-pairing interactions between the P-site C74 and C75 and the P loopof 23S rRNA on the left and the A-site C75 with the A loop of 23S rRNAon the right. The catalytic A2486 is near the phosphate oxygen (P) thatis the analogue of the tetrahedral intermediate oxyanion. FIG. 11(B)shows A2637 (in all blue) lying between the two CCA's and A2486 (green)whose N3 approaches a non-bridging phosphate oxygen. The N1 atoms of theA76 bases from the A- and P-site tRNAs are making nearly identicalinteractions with a ribose 2′ OH in both the A- and P-loops,respectively, and an approximate 2-fold axis relates these residues.

FIG. 12 shows a space filling model of the 23S and 5S rRNA, the proteinsand the CCdA-p-Puro inhibitor viewed down the active site cleft in arotated “crown view.” The bases are white and the sugar phosphatebackbones are yellow. The inhibitor is shown in red and the numberedproteins are shown in blue. The L1 and L11 proteins positioned at lowerresolution are in blue backbone. The central protuberance is labeled CP.

FIG. 13(A) shows a stereo view diagram of the three-dimensionaldistribution of the residues comprising the loops A and P and thepeptidyl transferase loop. FIG. 13(B) shows a stereo view of the centralloop in domain V from the direction of the tunnel. The residues arecolor coded based on mutations which confer antibiotic resistance. FIG.13(C) shows domain V active site with its central loop shown as thesecondary structure.

FIGS. 14(A) and (B) show the closest approach of polypeptides to thepeptidyl transferase active site marked by a ball and stickrepresentation of the Yarus inhibitor, CCdA-p-Puro. Specifically, FIG.14(A) shows a coil representation of domain V RNA backbone in red andbases in gray and a ribbon backbone representation of all thirteenproteins that interact with it. FIG. 14(B) shows a close-up view of theactive site with the RNA removed. The phosphate of the Yarus analogueand the proteins whose extensions are closest to the inhibitor are shownin ribbon with their closest side-chains in all atom representation. Thedistances in A between the closest protein atoms and the phosphorousanalogue of the tetrahedral carbon (pink) are shown, as is a modeledpeptide (pink).

FIG. 15 shows conserved nucleotides in the peptidyl transferase regionthat binds CCdA-p-Puro A space filling representation of the active siteregion with the Yarus inhibitor viewed down the active site cleft. Allatoms belonging to 23S rRNA nucleotides that are 95% conserved in allthree kingdoms (Gutell et al. (2000) supra) are colored red and allother nucleotides are white; the inhibitor is colored blue.

FIGS. 16(A)-(C) show the catalytic apparatus of the peptidyl transferaseactive site. Specifically, FIG. 16(A) shows stereo view of a portion ofthe experimental 2.4 Å resolution electron density map (Ban et al.(2000) Science 289: 905-920) of the large subunit in the region of thecatalytic site in stereo. The structure the RNA involved in interactionswith A2486 is superimposed. Residues G2102 (2061) and G2482 (2447) arehydrogen bonded to the N6 of A2486 (2451) and G2482 which interacts witha neighboring phosphate group. FIG. 16(B) shows a skeletalrepresentation with dashed hydrogen-bonds showing G2482, G2102, A2486and the buried phosphate that is proposed to result in a charge relaythrough G2482 to the N3 of A2486. FIG. 16(C) shows the normal and rarerimine tautomeric forms of G2482 and A2486 that are proposed to bestabilized by the buried phosphate of residue 2485.

FIGS. 17(A)-(C) show the proposed mechanism of peptide synthesiscatalyzed by the ribosome. Specifically, FIG. 17(A) shows the N3 ofA2486 abstracting a proton from the NH₂ group as the latter attacks thecarbonyl carbon of the peptidyl-tRNA. FIG. 17(B) shows a protonated N3stabilizing the tetrahedral carbon intermediate by hydrogen bonding tothe oxyanion. FIG. 17(C) shows the proton transferred from the N3 to thepeptidyl tRNA 3′ OH as the newly formed peptide deacylates.

FIGS. 18(A) and (B) show space filling representations of the 50Sribosomal subunit with the 3 tRNA molecules, in the same relativeorientation that they are found in the 70S ribosome structure by Nollerand colleagues docked onto the CCA's bound in the A-Site and P-Site.Specifically, FIG. 18(A), shown on the left-hand side, shows the wholesubunit in rotated crown view with the rRNA in yellow, proteins in pinkand tRNAs in orange. FIG. 18(B), shown on the right-hand side, shows aclose-up view showing the numbered proteins are in pink and the rRNA inblue. A backbone ribbon representation of the A-, P-, and E-sites areshown in yellow, red and white, respectively.

FIGS. 19(A)-(F) show the polypeptide exit tunnel. Specifically, FIG.19(A) shows the subunit cut in half, roughly bisecting its centralprotuberance and its peptide tunnel along the entire length. The twohalves have been opened like the pages of a book. All ribosome atoms areshown in CPK representation, with all RNA atoms that do not contactsolvent shown in white and all protein atoms that do not contact solventshown in green. Surface atoms of both protein and RNA are color-codedwith carbon in yellow, oxygen in red, and nitrogen in blue. A possibletrajectory for a polypeptide passing through the tunnel is shown as awhite ribbon. The peptidyl transferase site (PT) is also shown. FIG.19(B) shows detail of the polypeptide exit tunnel with the distributionof polar and non-polar groups, with atoms colored as in FIG. 19(A), theconstriction in the tunnel formed by proteins L22 and L4 (green patchesclose to PT), and the relatively wide exit of the tunnel. A modeledpolypeptide is in white. FIG. 19(C) shows the tunnel surface withbackbone atoms of the RNA color coded by domain: domain I (white), II(light blue), III (gold), IV (green), V (orange), 5S (pink) and proteinsare blue. The peptidyl transferase center (PTC) is shown. FIG. 19(D) isa space filling representation of the large subunit surface at thetunnel exit showing the arrangement of proteins, some of which mightplay roles in protein secretion. The RNA is in white (bases) and yellow(backbone) and the numbered proteins are blue. A modeled polypeptide isexiting the tunnel in red. FIG. 19(E) shows a close-up view of the halfof the exit tunnel showing the relationship of the peptidyl transferasecenter (PTC) to proteins L4 (yellow) and L22 (blue). The Yarus inhibitorand a modeled peptide are purple and the 23S rRNA is in red and white.FIG. 19(F) shows a secondary structure schematic of 23S rRNA identifyingthe sequences that contact the tunnel in red.

FIG. 20 is a pictorial representation showing the spatial relationshipbetween the antibiotic anisomycin bound to a large ribosomal subunit.

FIG. 21 is a pictorial representation showing the spatial relationshipbetween the antibiotic blasticidin bound to a large ribosomal subunit.

FIG. 22 is a pictorial representation showing the spatial relationshipbetween the antibiotics carbomycin and tylosin bound to a largeribosomal subunit.

FIG. 23 is a pictorial representation showing the spatial relationshipbetween the antibiotic sparsomycin bound to a large ribosomal subunit.

FIG. 24 is a pictorial representation showing the spatial relationshipbetween the antibiotics virginiamycin M (streptogramin A) and carbomycinA bound to a large ribosomal subunit.

FIG. 25 is a pictorial representation showing the spatial relationshipbetween the antibiotic spiramycin bound to a large ribosomal subunit.

FIG. 26 is a pictorial representation showing the spatial relationshipbetween the antibiotic azithromycin bound to a large ribosomal subunit.

FIG. 27 is a pictorial representation showing the spatial relationshipbetween the antibiotic linezolid bound to a large ribosomal subunit.

FIG. 28 is a pictorial representation showing the spatial relationshipbetween the antibiotic erythromycin bound to a large ribosomal subunit.

FIG. 29 is a pictorial representation showing the spatial relationshipof certain antibiotics, namely, anisomycin, blasticidin, carbomycin A,and virginiamycin M, bound to a large ribosomal subunit. The locationsof the bound antibiotics are shown relative to the ribosomal A-site,P-site, and polypeptide exit tunnel.

FIGS. 30(A)-(C) are pictorial representations showing a peptidyltransferase site disposed within a large ribosomal subunit. FIG. 30(A)shows a bound tylosin molecule, and identifies a disaccharide bindingpocket and two cavities denoted “cavity 1” and “cavity 2.” FIGS. 30(B)and (C) are provided on the left hand side to orient the reader to thelocations of the peptidyl transferase site (PT) and polypeptide exittunnel in the large ribosomal subunit.

FIG. 31 is a schematic representation of a computer system useful inmolecular modeling a ribosomal subunit and/or for performing rationaldrug design.

FIG. 32 is a schematic representation of certain potential drug targetsites in a large ribosomal subunit.

FIGS. 33(A)-(D) are pictorial representations showing the residueswithin the wall of the polypeptide exit tunnel that are conserved (red)or non-conserved (blue) between E. coli and rat. The ribosomal subunithas been sliced down the polypeptide exit tunnel with one half of thepolypeptide exit tunnel shown in FIG. 33(A), and the other half of thepolypeptide exit tunnel is shown in FIG. 33(B). FIG. 33(C) is providedto orient the reader to show the location of the portion of theribosomal subunit shown in FIG. 33(A) relative to the ribosomal subunitas a whole. FIG. 33(D) is provided to orient the reader to show thelocation of the portion of the ribosomal subunit shown in FIG. 33(B)relative to the large ribosomal subunit as a whole.

FIG. 34 is a schematic representation showing the synthesis of thehybrid antibiotic sparsoanisomycin from the individual antibioticssparsomycin and anisomysin.

FIG. 35 is a schematic representation showing the synthesis of twohybrid antibiotics, sparsochloramphenicol hybrids A and B, from theindividual antibiotics sparsomycin and chloramphenicol.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “active site” refers to regions on a ribosomeor ribosomal subunit that are directly involved in protein synthesis,e.g., the peptidyl transferase site, the A-site, the P-site, thepolypeptide exit tunnel, the elongation factor binding site, and othersimilar sites.

As used herein, the terms “agent,” “ligand” and “lead candidate” areused synonymously and refer to any atom, molecule, or chemical groupwhich binds or interacts with a ribosome, ribosomal subunit or ribosomefragment. Thus, ligands include, but are not limited to, a single heavyatom, an antibiotic or an analogue thereof, a tRNA, a peptidyl tRNA, anaminoacyl tRNA, or a signal recognition particle (“SRP”).

As used herein, “archaebacteria” refers to the kingdom of monerans thatincludes methane producers, sulfur-dependent species, and many speciesthat tolerate very salty or hot environments.

As used herein, the term “A-site” refers to the locus occupied by anaminoacyl-tRNA molecule immediately prior to its participation in thepeptide-bond forming reaction.

As used herein, the term “asymmetric unit” refers to a minimal set ofatomic co-ordinates that when operated upon by the symmetry operationsof a crystal will regenerate the entire crystal.

As used herein, “at least a portion of” or “at least a portion of thethree-dimensional structure of” is understood to mean a portion of thethree-dimensional structure of a ribosome or ribosomal subunit,including charge distribution and hydrophilicity/hydrophobicitycharacteristics, formed by at least three, more preferably at leastthree to ten, and most preferably at least ten amino acid and/ornucleotide residues of the ribosome or ribosomal subunit. The residuesforming such a portion may be, for example, residues which form acontiguous portion of the primary sequence of a ribosomal RNA orribosomal protein, residues which form a contiguous portion of thethree-dimensional structure of the ribosome or ribosomal subunit, or acombination thereof. As used herein, the residues forming “a portion ofthe three-dimensional structure of” a ribosome or ribosomal subunit,form a three-dimensional shape in which each atom or functional groupforming the portion of the shape is separated from the nearest atom orfunctional group forming the portion of the shape by no more than 40 Å,preferably by no more than 20 Å, more preferably by no more than 5-10 Å,and most preferably by no more than 1-5 Å.

As used herein, the term“atomic co-ordinates” or “structureco-ordinates” refers to mathematical co-ordinates (represented as“X,”“Y” and“Z” values) that describe the positions of atoms in a crystal ofa ribosome or ribosomal subunit. The diffraction data obtained from thecrystals are used to calculate an electron density map of the repeatingunit of the crystal. The electron density maps are used to establish thepositions of the individual atoms within a single ribosomal subunit.Those of skill in the art understand that a set of structureco-ordinates determined by X-ray crystallography is not without standarderror. For the purpose of this invention, the structures of tworibosomes, ribosomal subunits or portions thereof are considered to bethe same if they satisfy one of the following two tests. In a firsttest, the structures are considered to be the same if a set of structureco-ordinates for a ribosome or ribosomal subunit from any source has aroot mean square deviation of non-hydrogen atoms of less than about 2.0Å, or more preferably less than about 0.75 Å, when superimposed on thenon-hydrogen atom positions of the atomic co-ordinates deposited at theResearch Collaboratory for Structural Bioinformatics (RCSB) Protein DataBank (PDB) (Berman et al. (2000) Nucleic Acids Research 28, 235-242; seealso. the web page at URL rcsb.org/pdb/) with the accession numbers PDBID: 1FFK; PDB ID: 1FFZ; PDB ID: 1FG0; PDB ID: 1JJ2;PDB ID: 1K73; PDB ID:1KC8; PDB ID: 1K8A; PDB ID: 1KD1; or PDB ID: 1K9M, or contained on Disk1 of 1, the disclosure of each of the foregoing of which is incorporatedherein by reference in its entirety. In a second test, the structuresare considered to be the same if the r.m.s. deviation between a set ofatoms in a test structure and a corresponding set of atoms in areference structure is less than 2.0 Å. For the purposes of this test,the set of atoms in the reference structure comprises at least five ofthe series of 23S rRNA residues listed below as 631-633, 835-841,844-846, 882-885, 1836-1839, 2095-2105, 2474-2478, 2485-2490, 2528-2530,2532-2543, 2607-2612, 2614-2623, 2642-2648 of the structure deposited inthe PDB under accession number PDB ID: 1JJ2 or contained as file name1JJ2.RTF on Disk 1 of 1. The residues in the test structurecorresponding to the ones listed above are identified by sequencealignment using the program Lasergene v. 5.0 (DNA Star, Inc., Madison,WI) with the default settings. Specifically, the computer program isused to align those residues listed above in the Haloarcula marismortui23 S rRNA sequence with those in the test organism's rRNA. Once aligned,the corresponding residues in the test organism's rRNA are identified.The atomic co-ordinates of backbone atoms (P, C5′, 05′, C4′, C3′, 03′)of atoms in the test structure are superimposed upon the correspondingbackbone atoms (P, C5′, 05′, C4′, C3′, 03′) of the reference structureusing the program MIDAS Plus (Ferrin et al. (1988) J. Mol. Graphics6:13-27 and 36-37). The test and reference structures are considered thesame if the r.m.s. deviation between the two sets of atoms aftersuperpositioning is less than 2.0 Å, as determined by MIDAS Plus.

In the list of atomic co-ordinates deposited at the RCSB Protein DataBank or included herein as files recorded on the compact disks, the term“atomic co-ordinate” or structure co-ordinates refer to the measuredposition of an atom in the structure in Protein Data Bank (PDB) format,including X, Y, Z and B, for each. The term “atom type” refers to theelement whose co-ordinates are measured. The first letter in the columndefines the element. The term “X”, “Y”, “Z” refers to thecrystallographically defined atomic position of the element measuredwith respect to the chosen crystallographic origin. The term “B” refersto a thermal factor that measures the mean variation of an atom'sposition with respect to its average position.

Reference is made to the sets of atomic co-ordinates and related tablesincluded with this specification and submitted on compact disk (twototal compact disks including one original compact disk, and aduplicative copy of original compact disks). Disk No. 1 containsthirty-nine files. Disk No 1 contains the files identified asPDB1FFK.DOC and PDB1FFK.ENT which represent files of co-ordinatesdefining the large ribosomal subunit; PDB1FFZ.DOC and PDB1FFZ.ENT whichrepresent files of the co-ordinates defining the large ribosomalsubunit—CCdA-p-Puro complex; and PDB1FGO.DOC and PDB1FGO.ENT whichrepresent files of the co-ordinates defining the large ribosomalsubunit—aa-tRNA analogue complex; 1JJ2.RTF and 1JJ2.TXT which representfiles of the co-ordinates defining the completely refined largeribosomal subunit; anisomycin.pdb, blasticidin.pdb, carbomycin.pdb,sparsomycin.pdb, spiramycin.pdb, tylosin.pdb and virginiamycin.pdb whichrepresent files of the co-ordinates defining the large ribosomal subunitbound to anisomycin, blasticidin, carbomycin, sparsomycin, spiramycin,tylosin, and virginiamycin, respectively; three folders: FOLDERAcontains the file identified as 1JJ2.PDB (which represents a file of amore highly refined co-ordinates defining the large ribosomal subunit),FOLDERB contains the files identified as ANISOMYC.PDB, BLASTICI.PDB,CARBOMYC.PDB, SPARSOMY.PDB, SPIRAMYC.PDB, TYLOSIN.PDB, and VIRGLNIA.PDB(which represent files of the refined co-ordinates defining the largeribosomal subunit bound to anisomycin, blasticidin, carbomycin,sparsomycin, spiramycin, tylosin, and virginiamycin, respectively),FOLDERC contains the files identified as AZITHROM.PDB, and LINEZOLI.PDB(which represent files of the co-ordinates defining the large ribosomalsubunit bound to azitbromycin and linezolid, respectively); the fileidentified as erythromycin.pdb (which represents a file of theco-ordinates defining the large ribosomal subunit bound toerythromycin), and azithromycin.pdb and linezolid.pdb (which representfiles of the refined co-ordinates defining the large ribosomal subunitbound to azithromycin and linezolid, respectively).

As will be apparent to those of ordinary skill in the art, the atomicstructures presented herein are independent of their orientation, andthat the atomic co-ordinates identified herein merely represent onepossible orientation of a particular large ribosomal subunit. It isapparent, therefore, that the atomic co-ordinates identified herein maybe mathematically rotated, translated, scaled, or a combination thereof,without changing the relative positions of atoms or features of therespective structure. Such mathematical manipulations are intended to beembraced herein.

As used herein, the terms “atomic co-ordinates derived from” and “atomsderived from” refers to atomic co-ordinates or atoms derived, eitherdirectly or indirectly, from an electron density map. It is understoodthat atomic co-ordinates or atoms derived “directly” from an electrondensity map refers to atomic co-ordinates or atoms that are identifiedfrom and/or fitted to an electron density map by using conventionalcrystallographic and/or molecular modeling techniques and thus can beconsidered to be primary atomic co-ordinates or atoms. It is understoodthat atomic co-ordinates or atoms derived “indirectly” from an electrondensity map refers to atomic co-ordinates or atoms that are derived fromand thus are derivatives or transforms of the primary atomicco-ordinates or atoms and thus can be considered to be secondary atomicco-ordinates or atoms. The secondary atomic co-ordinates or atoms may begenerated from the primary atomic co-ordinates or atoms by usingconventional molecular modeling techniques. By way of a non limitingexample, the atomic co-ordinates for the H. marismortui large ribosomalsubunit as described hereinbelow are considered to be primaryco-ordinates, whereas the atomic co-ordinates of a mammalian largeribosomal subunit which can be derived from H marismortui atomicco-ordinates by molecular modeling, including, for example, homologymodeling and/or molecular replacement, are considered to be secondaryco-ordinates. Both types of atomic co-ordinates and atoms are consideredto be embraced by the invention.

As used herein the terms “bind,” “binding,” “bound,” “bond,” or“bonded,” when used in reference to the association of atoms, molecules,or chemical groups, refer to any physical contact or association of twoor more atoms, molecules, or chemical groups (e.g., the binding of aligand with a ribosomal subunit refers to the physical contact betweenthe ligand and the ribosomal subunit). Such contacts and associationsinclude covalent and non-covalent types of interactions.

As used herein, the terms “complex” or “complexed” refer to the assemblyof two or more molecules to yield a higher order structure, such as, a50S ribosomal subunit bound to a ligand.

As used herein, the term “computational chemistry” refers tocalculations of the physical and chemical properties of the molecules.

As used herein, the term “conjugated system” refers to more than twodouble bonds that are positioned spatially so that their electrons arecompletely delocalized with the entire system. Aromatic residues containconjugated double bond systems.

As used herein, the terms “covalent bond” or “valence bond” refer to achemical bond between two atoms in a molecule created by the sharing ofelectrons, usually in pairs, by the bonded atoms.

As used herein, the term “crystal” refers to any three-dimensionalordered array of molecules that diffracts X-rays.

As used herein, the term “crystallographic origin” refers to a referencepoint in the unit cell with respect to the crystallographic symmetryoperation.

As used herein, the term “elongation factor binding domain” refers tothe region of the ribosome that interacts directly with elongationfactors, including, for example, the elongation factors, EF-Tu and EF-G.

As used herein, the term “E-site” refers to the locus occupied by adeacylated tRNA molecule as it leaves the ribosome following itsparticipation in peptide-bond formation.

As used herein, the term “heavy atom derivatization” refers to themethod of producing a chemically modified form, also known as a “heavyatom derivative”, of a crystal of the ribosome and the ribosomal subunitand its complexes. In practice, a crystal is soaked in a solutioncontaining heavy metal atom salts, or organometallic compounds, e.g.,mercury chlorides, ethyl-mercury phosphate, osmium pentamine, or iridiumpentamine, which can diffuse through the crystal and bind to theribosome or ribosomal subunit. The location(s) of the bound heavy metalatom(s) can be determined by X-ray diffraction analysis of the soakedcrystal. This information, in turn, is used to generate the phaseinformation used to construct three-dimensional structure of the complex(Blundell et al. (1976) supra).

As used herein, the term“homologue” is understood to mean any one orcombination of (i) any protein isolated or isolatable from a ribosome ora ribosomal subunit (i.e., a ribosomal protein), (ii) any nucleic acidsequence isolated or isolatable from a ribosome or ribosomal subunit(i.e., a ribosomal RNA), (iii) any protein having at least 25% sequenceidentity to a ribosomal protein isolated from E. coli or Rattusnorvegicus as determined using the computer program“BLAST” versionnumber 2.1.1 implementing all default parameters, or (iv) any nucleicacid having at least 30% sequence identity to a ribosomal RNA isolatedfrom E. coli or Rattus norvegicus as determined using the computerprogram“BLAST” version number 2.1.1 implementing all defaultparameters.“BLAST” version number 2.1.1 is available and accessible viathe world wide web at the URL ncbi.nlm.nih.gov/BLAST/ or can be runlocally as a fully executable program on a standalone computer.

As used herein, the term “homology modeling” refers to the practice ofderiving models for three-dimensional structures of macromolecules fromexisting three-dimensional structures for their homologues. Homologymodels are obtained using computer programs that make it possible toalter the identity of residues at positions where the sequence of themolecule of interest is not the same as that of the molecule of knownstructure.

As used herein, the term “hydrogen bond” refers to two electronegativeatoms (either O or N), which share a hydrogen that is covalently bondedto only one atom, while interacting with the other.

As used herein, the term “hydrophobic interaction” refers tointeractions made by two hydrophobic residues.

As used herein, the terms “IC₅₀” or “inhibitory concentration ₅₀” areunderstood to mean the concentration of a molecule that inhibits 50% ofthe activity of a biological process of interest, including, withoutlimitation, cell viability and/or protein translation activity.

As referred to herein, ribosomal proteins are designated “LX” or “SX”,where L stands for “large subunit; S stands for “small subunit”; and Xin either case is an integer.

As used herein, the term “MIR” refers to multiple isomorphousreplacement, a technique used for deriving phase information fromcrystals treated with heavy atom compounds.

As used herein, the term “molecular graphics” refers tothree-dimensional representations of atoms, preferably on a computerscreen.

As used herein, the terms “molecular model” or “molecular structure”refer to the three-dimensional arrangement of atoms within a particularobject (e.g. the three-dimensional structure of the atoms that comprisea ribosome or ribosomal subunit, and the atoms that comprise a ligandthat interacts with a ribosome or ribosomal subunit, particularly with alarge ribosomal subunit, more particularly with a 50S ribosomalsubunit).

As used herein, the term “molecular modeling” refers to a method orprocedure that can be performed with or without a computer to make oneor more models, and, optionally, to make predictions about structureactivity relationships of ligands. The methods used in molecularmodeling range from molecular graphics to computational chemistry.

As used herein, the term “molecular replacement” refers to a method thatinvolves generating a model of a ribosome or ribosomal subunit whoseatomic co-ordinates are unknown, by orienting and positioning the atomicco-ordinates described in the present invention in the unit cell of thecrystals of the unknown ribosome so as best to account for the observeddiffraction pattern of the unknown crystal. Phases can then becalculated from this model and combined with the observed amplitudes togive the atomic co-ordinates of the unknown ribosome or ribosomalsubunit. This type of method is described, for example, in The MolecularReplacement Method, (Rossmann, M. G., ed.), Gordon & Breach, New York,(1972).

As used herein, “noncovalent bond” refers to an interaction betweenatoms and/or molecules that does not involve the formation of a covalentbond between them.

As used herein, the term “peptidyl transferase site” refers to the locusin the large ribosomal subunit where peptide bonds are synthesized.

As used herein, the term “polypeptide exit tunnel” refers to the channelthat passes through the large ribosomal subunit from the peptidyltransferase site to the exterior of the ribosome through which newlysynthesized polypeptides pass.

As used herein, the term “protein synthesis inhibitor” refers to anymolecule that can reduce, inhibit or otherwise disrupt protein orpolypeptide synthesis in a ribosome.

As used herein, the term “P-site” refers to the locus occupied by apeptidyl-tRNA at the time it participates in the peptide-bond formingreaction.

As used herein, the term “ribofunctional locus” refers to a region ofthe ribosome or ribosomal subunit that participates, either actively orpassively, in protein or polypeptide synthesis within the ribosome orribosomal subunit and/or export or translocation of a protein orpolypeptide out of a ribosome. The ribofunctional locus can include, forexample, a portion of a peptidyl transferase site, an A-site, a P-site,an E-site, an elongation factor binding domain, a polypeptide exittunnel, and a signal recognition particle (SRP) binding domain. It isunderstood that the ribofunctional locus will not only have a certaintopology but also a particular surface chemistry defined by atoms that,for example, participate in hydrogen bonding (for example, proton donorsand/or acceptors), have specific electrostatic properties and/orhydrophilic or hydrophobic character.

As used herein, the term “ribosomal subunit” refers to one of the twosubunits of the ribosome that can function independently during theinitiation phase of protein synthesis but which both together constitutea ribosome. For example, a prokaryotic ribosome comprises a 50S subunit(large subunit) and a 30S subunit (small subunit).

As used herein, the term “ribosome” refers to a complex comprising alarge ribosomal subunit and a small ribosomal subunit.

As used herein, the term “signal recognition particle binding domain”refers to the portion of the ribosome that interacts directly with thesignal recognition particle.

As used herein, the term “space group” refers to the arrangement ofsymmetry elements of a crystal.

As used herein, the term “symmetry operation” refers to an operation inthe given space group that places the atoms in one asymmetric unit onthe corresponding atoms in another asymmetric unit.

As used herein, the term “twinned” refers to a single macroscopiccrystal that contains microscopic domains of the same symmetry thatdiffer significantly in orientation in such a way that the diffractionpatterns of all are superimposed. In a twinned crystal the mosaicblocks, or domains, are orientated so that some point in one directionand others point in a second, distinctly different direction, and thedirections are such that the diffraction pattern generated by one groupof blocks falls exactly on top of the diffraction pattern of the othergroup.

As used herein, the term “untwinned” refers to a crystal cell thedomains of which are aligned. The domains are also known as the “mosaicblocks.” Most crystals diffract as though they were assemblies of mosaicblocks. One can think of them as small, perfectly ordered regions withinthe larger crystal, which, overall, is not so well ordered. Each blockhas the same symmetry and unit cell packing as all the others.

As used herein, the term “unit cell” refers to a basic parallelepipedshaped block. The entire volume of crystal may be constructed by regularassembly of such blocks. Each unit cell comprises a completerepresentation of the unit of pattern, the repetition of which builds upthe crystal.

II. Structure and Use of the Large Ribosomal Subunit

A. Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution,Initial Refinement

The present invention is based, in part, on the development of a novelmethod for preparing crystals of ribosomes. The novel method providescrystals of the 50S ribosomal subunit that are much thicker than thoseavailable earlier and that can diffract X-rays to a resolution of about2.4 Å. The method eliminates the twinning of crystals that obstructedprogress in determining the crystal structure of the 50S ribosomalsubunit from H. marismortui for many years. The method of preparing thecrystals of the 50S ribosomal subunit is discussed below.

The present invention is also based, in part, on the atomic structure ofthe crystal of the 50S ribosomal subunit from H. marismortul that hasbeen derived from a 2.4 Å resolution electron density map that wasexperimentally phased using heavy atom derivatives. The atomicco-ordinates defining the large ribosomal unit were deposited on Jul.10, 2000, at Research Collaboratory for Structural Bioinformatics (RCSB)Protein Data Bank (PDB) (Berman et al. (2000) Nucleic Acid Research 28,235-242; see also, the web page at the URL rcsb.org/pdb/) with accessionnumber PDB ID: 1FEK.

Moreover, the present invention is based, in part, on the derivationfrom the atomic co-ordinates of the following model which is brieflysummarized here and discussed in detail in the following sections of thespecification. This model includes 2,811 of the 2,923 nucleotides of 23SrRNA, all 122 nucleotides of its 5S rRNA, and structures for the 27proteins that are well-ordered in the subunit.

The secondary structures of both 5S and 23S rRNA are remarkably close tothose deduced for them by phylogenetic comparison. The secondarystructure of the 23S rRNA divides it into 6 large domains, each of whichhas a highly asymmetric tertiary structure. The irregularities of theirshapes notwithstanding, the domains fit together in an interlockingmanner to yield a compact mass of RNA that is almost isometric. Theproteins are dispersed throughout the structure, concentrated largely onits surface, but they are much less abundant in the regions of thesubunit that are of primary functional significance to proteinsyntheses—the 30S subunit interface, the binding regions for tRNA andthe peptidyl transferase active site. The most surprising feature ofmany of these proteins are the extended, irregular structures of theirloops and termini, which penetrate between RNA helices. The primary roleof most of the proteins in the subunit appears to be stabilization ofthe three-dimensional structure of its rRNA.

1. Preparation of the Crystal for the 50S Ribosomal Subunit andStructure Determination.

Several experimental approaches were used to extend the resolution ofthe electron density maps of the H. marismortui 50S ribosomal subunitfrom 5 Å to 2.4 Å including improvements in the crystals. Aback-extraction procedure was developed for reproducibly growingcrystals that are much thicker than those available earlier and candiffract to 2.2 Å resolution (see, Example 1). Briefly, the crystalswere grown at room temperature in hanging drops by vapor diffusion fromseeded solutions back-extracted from precipitated subunits. The crystalsthat resulted had maximum dimensions of 0.5×0.5×0.2 mm and wereharvested after three weeks. The twinning of crystals that obstructedprogress for many years (Ban et al. (1999) supra) was eliminated byadjusting crystal stabilization conditions (see, Example 1). Crystalswere stabilized by gradual transfer into a solution containing 12%, PEG6000, 22% ethylene glycol, 1.7 M NaCl, 0.5 M NH₄Cl, 100 mM potassiumacetate, 30 mM MgCl₂ and 1 mM CdCl₂, pH 6.2, and flash frozen in liquidpropane. Reducing the salt concentration below 1.7 M NaCl (KCl)increased the tendency of crystals to become twinned. At saltconcentrations as low as 1.2 M nearly all of the crystals were twinned.

All the X-ray data used for high resolution phasing were collected atthe Brookhaven National Synchrotron Light Source except for two nativedata sets used, which were collected at the Advanced Photon Source atArgonne (see, Example 2) (Table 1). Osmium pentamine (132 sites) andIridium hexamine (84 sites) derivatives proved to be the most effectivein producing both isomorphous replacement and anomalous scattering phaseinformation to 3.2 Å resolution (see, Example 2). Inter-crystal densityaveraging which had contributed significantly at lower resolution, wasnot helpful beyond about 5 Å resolution. Electron density maps weredramatically improved and their resolutions extended, eventually to 2.4Å, using the solvent flipping procedure in CNS (Abrahams et al. (1996)Acta Crystollogr. D 52: 30; Brünger et al. (1998) Acta Crystallogr. DBiol. Crystallogr. 54: 905-921).

TABLE 1 Statistics for Data Collection, Phase Determination, and ModelConstruction Data Statistics MIRAS1 MIRAS2 Native1 Os(NH₃)₅ ²⁺ UO₂F₅ ³⁻Native2 Ir(NH₃)₆ ³⁺ Os(NH₃)₆ ³⁺ Ta₆Br₁₂ ²⁺ Heavy atom — 30.0 0.5 — 20.04.5 3.0 conc. (mM) Soaking time (hrs) — 1.5 4 — 24 hrs 24 hrs 24 hrsSites no. — 132 20 — 84 38 9 Resolution (Å) 90-2.4 40-3.5 40-3.8 30-2.930-3.2 30-3.5 30-3.8 (*) (2.5-2.4) (3.6-3.5) (3.9-3.8) (3.0-2.9)(3.32-3.22) (3.27-3.20) (3.6-3.5) (3.97-3.80) λ(Å) 1.00 1.14 1.30 1.001.075 1.14 1.255 Observations 6,089,802 1,308,703 596,166 2,832,3601,823,861 1,646,468 1,288,524 Unique 665,928 429,761 313,863 390,770541,488 488,275 346,745 Redun. (*) 9.1 (6.5) 3.0 (2.5) 1.9 (1.6) 7.2 3.44.3 (4.2) 3.7 Completeness(*) 95.6 (71.0) 99.4 (96.8) 92.0 (54.1) 97.193.8 98.1 (99.0) 99.5 I/σI (Last bin) 25.5 (1.9) 13.5 (3.3) 8.9 (1.6)18.0 (6.4) 12.0 (2.6) 10.6 (2.7) 10.8 (3.2) R_(merge) (*) 8.6 (69.1) 7.2(32.0) 9.1 (37.9) 11.2 (36.9) 8.5 (29.5) 12.1 (46.0) 12.1 (40.5) 02(ano) (*) — 2.8 (1.0) 1.5 (1.0) — 2.63 (1.48) 1.8 (1.0) 2.42 (1.18)R_(merge) (ano) — 6.2 8.0 — 6.7 6.9 R_(iso) — 14.1 (22.7) 26.4 (47.0) —12.9 (28.1) 19.5 (39.4) Phasing Statistics Resolution shells (Å): ˜73,200 reflections per bin 30.0 5.1 4.0 3.5 3.2 Total MIRAS1 (FOM) 0.520.31 0.14 — 0.32 Os(NH₃)₅ ²⁺ Phasing power 0.87 0.72 0.66 — 0.75 Phasingpower (SAD) 1.40 0.58 0.26 — 0.75 R_(cullis) (centric) 0.62 0.65 0.67 —0.65 UO₂F₅ ³⁻ Phasing Power 0.47 0.33 0.28 — 0.36 Phasing power (SAD)0.46 0.25 — — 0.36 R_(cullis) (centric) 0.72 0.77 0.75 — 0.75 MIRAS2(FOM) 0.48 0.40 0.28 0.12 0.33 Ir(NH₃)₆ ³⁺ Phasing power 1.02 0.92 0.780.66 0.89 Phasing power (SAD) 2.02 1.60 1.22 0.83 1.47 R_(cullis)(centric) 0.58 0.63 0.70 0 74 0.63 Os(NH₃)₆ ³⁺ Phasing power 0.62 0.570.58 0.58 0.59 Phasing power (SAD) 0.47 0.39 — — 0.42 R_(cullis)(centric) 0.78 0.78 0.78 0.76 0.78 Ta₆Br₁₂ ²⁺ (Used for SAD phasingonly) Phasing Power (SAD) 2.77 0.35 0.13 — 119 FOM_((MIRAS1+MIRAS2+SAD)) 0.76 0.51 0.31 0.14 0.37 Model StatisticsResolution range (Å) 90.0-2.4 rms deviations Average B factors (Å²)Reflections 577,304 Bonds (Å) 0.0064 All atoms 37.4 R_(sryst) (%) 25.2Angles (°) 1.19 23S rRNA 32.3 R_(free) (%) 26.1 Dihedrals (°) 28.8 5SrRNA 43.2 Impropers (°) 1.68 Minimum/Max B factors (Å²) 70/107.9 λ,wavelength; Redun., redundancy; (*) last-resolution shell. R_(iso):Σ|F_(PH)-F_(P)|/ΣF_(PH), where F_(PH) and F_(P) are the derivative andthe native structure factor amplitudes, respectively. R_(sym):ΣΣ_(I)|I_((h))-I_((h)1)|/ΣΣ:I_((h)1), where I(h) is the mean intensityafter reflections. Phasing power: r.m.s. isomorphous difference dividedby the r.m.s. residual lack of closure. R_(cullis):Σ(∥F_(PH)-F_(P|)-_(|)F_(H(calc))∥)/Σ_(|)F_(PH)-F_(p)|, where F_(PH) isthe structure factor of the derivative and F_(P) is that of the nativedata. The summation is valid only for centric reflection. FOM (figure ofmerit): mean value of the cosine of the error in phase angles.Abbreviations: MIRAS: multiple isomorphous replacement, anomalousscattering; SAD: single wavelength anomalous diffraction; FOM: figure ofmerit. Except for regions obscured by disorder, theexperimentally-phased, 2.4 Åresolution electron density map was ofsufficient quality so that both protein and nucleic acid sequencingerrors could be identified and corrected. Each nucleotide could befitted individually and the difference between A and G was usually clear# without reference to the chemical sequence, as was the distinctionbetween purines and pyrimidines (FIG. 1).

Subtraction of the atomic model from the experimental electron densitymap leaves no significant density except for water and ions, showingthat the model accounts for all the macromolecular density. Preliminaryrefinement of the model was achieved using a mixed target in the programCNS (Brünger et al. (1998) supra). The model was further refined in realspace against the 2.4 Å electron density map using the program TNT(Tronrud (1997), Macromolecular Crystallography, Part B, Methods InEnzymology), which yielded a model with a free R-factor of 0.33. Oneadditional round of mixed target refinement of both atomic positions andB-factors using CNS led to the structure described below. Its freeR-factor is 0.27 (Table 1).

2. Sequence Fitting and Protein Identification.

The sequence of 23S rRNA was fit into the electron density mapnucleotide by nucleotide starting from its sarcin/ricin loop sequence(A2691-A2702) (E. coli numbers A2654 to A2665), whose position had beendetermined at 5 Å resolution (Ban et al. (1999) supra). Guided by theinformation available about the secondary structures of 23S rRNAs(Gutell, R. R. (1996), “Comparative Sequence Analysis and the Structureof 16S and 23S rRNA,” Ribosomal RNA. Structure, Evolution, Processing,and Function in Protein Biosynthesis, (Dahlberg A. and Zimmerman B.,eds.), CRC Press, Boca Raton, Fla. pp. 111-128), the remaining RNAelectron density neatly accommodated the sequence of 5S rRNA. Theinterpretation of protein electron density corresponding to the proteinwas more complicated because each protein region had to be identifiedchemically before the appropriate sequence could be fit into it, butabout 4,000 amino acid residues were fit into electron density.

The H. marismortui 50S subunit appears to contain thirty-one proteins,and there are sequences in the Swiss-Prot data bank for twenty eight ofthose thirty one proteins, including one, HMS6 or L7ae, that wasoriginally assigned to the small ribosomal subunit (Whittmann-Liebold etal. (1990) supra). The three remaining proteins were identified usingthe sequences of the ribosomal proteins from eukaryotes and otherarcheal species as guides. No electron density was found for one of theH. marismortui large ribosomal subunit proteins in the sequencedatabase, LX. Either the assignment of LX to the large subunit is inerror, or LX is associated with a disordered region of the subunit, orLX is absent from the subunits examined altogether.

The 2.4 Å resolution electron density map lacks clear electron densityfor proteins L1, L10, L11, and L12, the positions of which are knownfrom earlier low resolution X-ray and/or electron microscopic studies.These proteins are components of the two lateral protuberances of thesubunit, which are both poorly ordered in these crystals. L1 is the soleprotein component of one of them (Oakes, M. et al. (1986)), Structure,Function and Genetics of Ribosomes, (Hardesty, B. and Kramer, G., eds.)Springer-Verlag, New York, N.Y., 47-67) and is evident in 9 Å resolutiondensity maps of the subunit (Ban et al. (1998) supra), but not at higherresolutions. L10, L11 and L12 are components of the other protuberance,which is often referred to as the L7/L12 “stalk” (Oakes et al. (1986)supra). L11 and the RNA to which it binds were located in the 5 Åresolution electron density map of the H. marismortui large subunit (Banet al. (1999) supra) using the independently determined crystalstructures of that complex (Conn GL et al. (1999) Science 284:1171-1174; Wimberly et al. (1999) Cell 97: 491-502). A protein fragment(about 100 residues) that is associated with the RNA stalk that supportsthe L11 complex can be seen in the 2.4 Å resolution map. Based onlocation, it must be part of L10. There is no electron densitycorresponding to L12 seen at any resolution, but the L12 tetramer isknown to be attached to the ribosome through L10, and the L10/L12assembly is known to be flexible under some circumstances (Moller et al.(1986) Structure, Function, and Genetics of Ribosomes, supra, pp.309-325), which may explain its invisibility here.

The structures of eubacterial homologues of proteins L2, L4, L6, L14,and L22 have previously been determined in whole or in part (see, Table2). L2, L6 and L14 were initially located in the 5 Å resolution map (Banet al. (1999) supra). L4 and L22 have now been identified and positionedthe same way. Electron density corresponding to most of the remainingproteins was assigned by comparing chain lengths and sequence motifsdeduced from the electron density map with known sequence lengths,guided by the information available about relative protein positions(Walleczek et al. (1988) EMBO J. 7: 3571-3576) and protein interactionswith 23S rRNA and 5S rRNA (Ostergaard et al. (1998) J. Mol. Biol. 284:227-240). Each of the protein electron density regions so identified iswell accounted for by its amino acid sequence.

The most interesting of the proteins identified by sequence similaritywas L7ae, which first appeared to be L30e. The L30e identificationseemed plausible because the structure of yeast L30 superimposes neatlyon the electron density of L7ae, and the structure of the RNA to whichL7ae binds closely resembles that of the RNA to which yeast L30 binds(Mao, H. et al. (1999) Nat. Struct. Biol. 6: 1139-1147). Nevertheless,the sequence of HMS6, which by sequence similarity is a member of theL7ae protein family, fits the electron density better. Four of the otherproteins identified by sequence similarity, L24e, L37e, L37ae, and L44e,contain zinc finger motifs. The rat homologues of L37e and L37ae werepredicted to be zinc finger proteins on the basis of their sequences(Wool et al. (1995) supra), and this prediction helped identify theirhomologues in H. marismortui.

TABLE 2 Large Subunit Proteins from Haloarcula marismortui InteractionsName¹ Hmlg² Lgth³ Conf⁴ 1 2 3 4 5 6 5S Proteins L1* 211 glb. + none L2†RL8 239 glb + ext + + + + (L37ae) L3 RL3 337 glb + ext + + + + L14,L24e, (L13) L4† RL4 246 glb + ext + + Å (Ll8c), (L24), (L37e) L5 RL11176 glb + + L18 L6 RL9 177 glb Å Å + (L13) L10* RP0 348 glb? + L12 L11*RL12 161 glb + none L12* RL1/2 115 glb L10 L13 RL13a 145 glb + Å Å (L3),(L6) L14 RL23 132 glb + + + L3, L24e L15 RL27a 164 glb + ext + + +(L18e), (L32e) L18 RL5 186 glb + ext Å + + L5, L21e L19 RL19 148 glb +ext + + + Å none L22 RL17 154 glb + ext + Å + + + + none L23 RL23a 84glb Å + L29, (L39e) L24 RL26 119 glb + ext + (L4) L29 RL35 70 glb + L23L30 RL7 154 glb + + none L18e RL18 115 glb + (L4), (L15) L21e RL21 95glb + + Å L18 L24e RL24 66 glb Å + L3, L14 L31e RL31 91 glb + + + noneL32e RL32 240 glb Å + (L15) L37e RL37 56 glb + ext + + + Å (L4) L39eRL39 49 ext + + (L23) L44e RL36a 92 glb + ext + Å + (L15e) L7ae R17a 110glb Å L15e L10e RL10 163 glb + + Å none L15e RL15 184 glb + ext + Å ÅÅ + (L44e), L7ae L37ae RL37a 72 glb + ext + + + L2 The top block ofproteins include all those known to have eubacterial homologues of thesame name. The second block lists proteins found in the H. marismortuilarge ribosomal subunit that have only eukaryotic homologues(Wittmann-Liebold et al. (1990) supra). Their names are all followed bythe letter “e” to distinguish them from eubacterial proteins that wouldotherwise have the same name. The third block are large subunit proteinsfor which no H. marismortui sequence yet exists. # They are identifiedby sequence homology using standard L names. ¹The structures of all orpart of homologues of the following proteins were previously determined:L1 (Nevskaya et al (2000) Struct. Fold. Des. 8: 363), L2 (Nakagawa, A.et al. (1999) EMBO J. 18: 1459-1467), L4 (Wahl et al. (2000) EMBO J. 19:807-818), L6 (Golden et al. (1993) EMBO J. 12: 4901-4908), L11 (Conn etal. (1999) supra; Wimberly et al. (1999) supra; Markus et al. (1997)Nature Struct. Biol. 4: 70-77), L12 (Leijonmarck, M. et al. (1980)Nature 286: 824-827), # L14 (Davies et al. (1996) Structure 4: 55-66),L22 (Unge et al. (1998) Structure 6: 1577-1586), L30 (Wilson et al.(1986) Proc Nat. Acad. Sci. USA 83: 7251-7255). All other structures,except 10, have been newly determined in this study. ²Rat homologue. Ratequivalents to H marismortui proteins are from (Mao et al. (1999)supra). ³Sequence chain length. ⁴Conformation: glb = globular; ext =extension ⁵The protein interactions with the 6 domains of 23S rRNA, 5SrRNA and other proteins are specified. (+) implies that the interactionis substantial. (Å) implies a weak, tangential interaction. Proteinnames are shown in parentheses implies that the interactions are weak;otherwise, the interaction is substantial. *All entries so designateddescribe proteins that are not fully represented in the electron densitymaps described here. The summary information provided is derived fromliterature sources and is included here for completeness only. †Thestructure available for this protein in isolation does not include theextension(s) reported here.

3. General Appearance of the Subunit.

In its crown view (see FIG. 2), the large ribosomal subunit, which isabout 250 Å across, presents its surface that interacts with the smallsubunit to the viewer with the three projections that radiate from thatsurface pointed up. Although the protuberance that includes L1 is notvisible in the 2.4 Å resolution electron density map, the structure ofL1, which has been determined independently (Nikonov et al. (1996) EMBOJ. 15: 1350-1359), has been positioned approximately in lower resolutionmaps (Ban et al. (1998) supra) and is included here to orient thereader. It is evident that, except for its two lateral protuberances,the large ribosomal subunit is monolithic. There is no hint of adivision of its structure into topologically separate domains. Inaddition, partly because it lacks obvious domain substructure but alsobecause it is so large, it is impossible to comprehend looking at it asa whole. In order to convey to the reader a sense of how it is puttogether, the subunit must be dissected into its chemical components.

4. RNA Secondary Structure.

All the base pairs in H. marismortui 23S rRNA stabilized by at least twohydrogen bonds were identified using a computer program that searchedthe structure for hydrogen bond donors and acceptors separated by lessthan 3.2 Å. Bases linked by at least two such bonds were consideredpaired if the angle between their normals was less than 45°, and theangle between bonds and base normals was also less than 45°. Based onthe results of this analysis, a secondary structure diagram has beenprepared in the format standard for 23S/28S rRNAs (see, FIG. 3). Thesecondary structure predicted for this molecule by phylogeneticcomparison was remarkably accurate, but it did not find all of thetertiary pairings and failed to identify interactions involvingconserved bases. In addition to base pairs of nearly every type, the RNAcontains numerous examples of well-known secondary structure motifs suchas base triplets, tetraloops, and cross-strand purine stacks, but nodramatically new secondary structure motifs have been identified so far.

The secondary structure of this 23S rRNA consists of a central loop thatis closed by a terminal stem, from which 11 more or less complicatedstem/loops radiate. It is customary to describe the molecule asconsisting of 6 domains, and to number its helical stems sequentiallystarting from the 5′ end (see, FIG. 4) (Leffers et al. (1987) supra).The division of the molecule into domains shown in FIG. 4 deviates fromstandard practice with respect to helix 25, which is usually consideredto be part of domain I. It is placed in domain II because it interactsmore strongly with domain II than it does with the other elements ofdomain I.

There are five sequences longer than 10 nucleotides in 23S rRNA whosestructures cannot be determined from the 2.4 Å resolution map due todisorder. Altogether they account for 207 out of the 232 nucleotidesmissing from the final model. The disordered regions are: (1) all ofhelix 1, (2) the distal end of helix 38, (3) helix 43/44 to whichribosomal protein L1 binds, (4) the loop end of stem/loop 69, and (5)helix 76/77/78, which is the RNA structure to which L1 binds. Forcompleteness, these regions are included in FIG. 3 (in gray) with theirsecondary structures determined phylogenetically.

5. Overall Architecture of rRNA.

The six domains of 23S rRNA and 5S rRNA all have complicated, convolutedshapes that nevertheless fit together to produce a compact, monolithicRNA mass (see FIG. 4(A) and 4(B)). Thus despite the organization of itsRNAs at the secondary structure level, in three-dimensions, the largesubunit is a single, gigantic domain. In this respect, it is quitedifferent from the small subunit, which is a flatter object that is notat all monolithic. Even in low resolution electron micrographs the smallsubunit consists of three structural domains, each of which, it turnsout, contains one of the three secondary structure domain of its RNA(Noller et al. (1990) The Ribosome: Structure, Function, and Evolution,supra, pp. 73-92). This qualitative difference between the two subunitsmay reflect a requirement for conformational flexibility that is greaterfor the small subunit.

Domain I, which looks like a mushroom (see, FIG. 4(E)), lies in the backof the particle, behind and below the L1 region. The thin part of thedomain starts in the vicinity of domain VI, which is where its first andlast residues are located. Helices 1 and 25 span the particle in theback and then the domain expands into a larger, more globular structurebelow and behind the L1 region.

The largest of the six 23S rRNA domains, domain II, which accounts formost of the back of the particle, has three protrusions that reachtowards the subunit interface side of the particle (see, FIG. 4(F)). Oneof them (helix 42-44) is the RNA portion of the L7/L12 stalk, which isknown to interact with elongation factors, is not well-ordered in thesecrystals. The second domain II protrusion is helix 38, which is thelongest, unbranched stem in the particle. It starts in the back of theparticle, bends by about 90 degrees and protrudes towards the smallsubunit between domains V and 5S rRNA. The third region, helix 32-35.1,points directly towards the small subunit and its terminus, the loop ofstem/loop 34, interacts directly with the small ribosomal subunit(Culver et al. (1999) Science 285: 2133-2135). This loop emerges at thesubunit interface between domains III and IV.

Domain III is a compact globular domain that occupies the bottom leftregion of the subunit in the crown view (see, FIG. 4(G)). It looks likea four pointed star with the origin of the domain (stem/loop 48) andstem/loops 52, 57, and 58 forming the points. The most extensivecontacts of domain III are with domain II, but it also interacts withdomains I, IV and VI. Unlike all the other domains, domain III hardlyinteracts with domain V at all; the sole contact is a single van derWaals contact involving a single base from each domain.

Domain IV accounts for most of the interface surface of the 50S subunitthat contacts the 30S subunit (see, FIG. 4(H)). It forms a largediagonal patch of flat surface on that side of the subunit, and connectsto domains III and V in the back of the particle. Helices 67-71 are themost prominent feature of domain IV, and form the front rim of theactive site cleft, which is clearly visible at low resolution (see, FIG.2). This is one of the few regions of the 23S rRNA that is notextensively stabilized by ribosomal proteins. Helix 69 in the middle ofthis ridge interacts with the long penultimate stem of 16S rRNA in thesmall ribosomal subunit and can be viewed as a divider separating A-sitebound tRNAs from P-site bound tRNAs.

Domain V, which is sandwiched between domains IV and II in the middle ofthe subunit, is known to be intimately involved in the peptidyltransferase activity of the ribosome. Structurally the domain can bedivided into three regions (see, FIGS. 4(I) and 4(J)). The first startswith helix 75 and ultimately forms the binding site for protein L1. Thesecond, which consists of helices 80-88, forms the bulk of the centralprotuberance region, and is supported in the back by the 5S rRNA anddomain II. The third region, which includes helices 89-93, extendstowards domain VI and helps stabilize the elongation factor bindingregion of the ribosome.

The smallest domain in 23S rRNA, domain VI, which forms a large part ofthe surface of the subunit immediately below the L7/L12 stalk, resemblesa letter X with a horizontal bar at the bottom (see, FIG. 4(K)). Aninteresting region of this domain is the sarcin-ricin loop (SRL)(stem/loop 95), the structure of which has been extensively studied inisolation (Szewczak et al. (1995) J. Mol. Biol. 247: 81-98). The SRL isessential for factor binding, and ribosomes can be inactivated by thecleavage of single covalent bonds in this loop (Wool et al. (1992) TIBS17: 266-269). As suggested by nucleotide protection data, the majorgroove of this loop is exposed to solvent (Moazed et al. (1988) Nature334: 362-364), and its conformation is stabilized by proteins andthrough interaction with domain V that involves two bases on the minorgrove side. The nucleotides involved are A 2699 and G 2700 in domain VI,and A 2566 and G 2567 in domain V.

5S ribosomal RNA, which is effectively the seventh RNA domain in thesubunit, consists of three stems radiating out from a common junctioncalled loop A (see, FIG. 4(D)). In contrast to what is seen in thecrystal structure of fragment 1 from E. coli 5S rRNA (Correll et al.(1997) Cell 91: 705-712), the helix ⅔ arm of the molecule stacks on itshelix ⅘ arm,not helix 1 (see, FIG. 4(L)). This arrangement results froma contorted conformation of loop A residues that involves two stackedbase triples. Indeed, from the secondary structure point of view, theloopA-helix 2,3 arm of 5S rRNA is quite remarkable. Nowhere else in thesubunit is there a higher concentration of unusual pairings and ofconvoluted RNA secondary structure.

6. Sequence Conservation and Interactions in 23S rRNA.

While 23S/28S rRNAs contain many conserved sequences, they also varysubstantially in chain length. Shorter 23S/28S rRNAs are distinguishedfrom their longer homologues by the truncation of, or even theelimination of entire stem/loops, and by comparing sequences, one canidentify a minimal structure that is shared by all (Gerbi (1995)Ribosomal RNA: Structure, Evolution, Processing and Function in ProteinBiosynthesis, supra, pp. 77-88). The expansion sequences in the 23S rRNAof H. marismortui, i.e., the sequences it contains that are larger thanthe minimum, are shown in FIG. 5 in green. They are largely absent fromthe subunit interface surface of the particle, but they are abundant onits back surface, far from its active sites. This is consistent with lowresolution electron microscopic observations suggesting that the regionof the large subunit whose structure is most conserved is the surfacethat interacts with the small subunit (Dube et al. (1998)Structure 6:389-399).

There are two classes of conserved sequences in 23S rRNA. One containsresidues concentrated in the active site regions of the large subunit.The second class consists of much shorter sequences scattered throughoutthe particle (FIG. 5: red sequences). The SRL sequence in domain VI andthe cluster of conserved residues belonging to domain V that are locatedat the bottom of the peptidyl transferase cleft are members of the firstclass. They are conserved because they are essential for substratebinding, factor binding and catalytic activity. Most of the residues inthe second class of conserved residues are involved in the inter- andintra-domain interactions that stabilize the tertiary structure of 23SrRNA. Adenosines are disproportionately represented in this class. Thepredominance of A's among the conserved residues in rRNAs has beenpointed out in the past (Ware et al. (1983) Nucl. Acids. Res. 22:7795-7817).

In addition to its reliance on A-dependent motifs, the tertiarystructure of the domains of 23S rRNA and their relative positions arestabilized by familiar tertiary structure elements like RNA zippers andtetraloop/tetraloop receptor motifs (Moore, P. B. (1999) Annu. Rev.Biochem. 68: 287-300), and in many places, base pairs and triplesstabilize the interactions of sequences belonging to differentcomponents of the secondary structure of 23S rRNA.

Interestingly, 5S rRNA and 23S rRNA do not interact extensively witheach other. The few RNA/RNA interactions there are involve the backbonesof the helix ⅘ arm of 5S rRNA and the backbone of helix 38 of 23S rRNA.Most of the free energy and all of the specificity of 5S rRNA binding tothe large ribosomal subunit appears to depend on its extensiveinteractions with proteins that act as modeling clay sticking it to therest of ribosome.

7. Proteins in the 50S Ribosomal Subunit.

The structures of twenty seven proteins found in the large ribosomalsubunit of H. marismortui (Table 2) have been determined. Twenty-one ofthese protein structures have not been previously established for anyhomologues, and the structures of the six that do have homologues ofknown structure have been rebuilt into the electron density map usingtheir H. marismortui sequences. In addition, there are structuresavailable for homologues of H. marismortui L1, L11 and L12, which cannotbe visualized in the 2.4 Å resolution electron density map. Only thestructure of L10 is still unknown among the thirty one proteins known tobe present.

Not every one of these structures is complete. For example, an entiredomain of L5 is missing from the electron density, presumably because ofdisorder. L32e is also noteworthy in this regard. About twenty residuesfrom its N-terminus are not seen in the electron density map, and theelectron density map suggests that its C-terminal residue is covalentlybound to the most N-terminal of its visible residues.

Of the thirty large subunit ribosomal proteins whose structures areknown, 17 are globular proteins, similar in character to thousands whosestructures are in the Protein Data Bank (Table 2). The remainingthirteen proteins either have globular bodies with extensions protrudingfrom them (“glb+ext”) or are entirely extended (“ext”). Their extensionsoften lack obvious tertiary structure and in many regions are devoid ofsecondary structure as well (see FIG. 6). These extensions may explainwhy many ribosomal proteins have resisted crystallization in isolation.The exceptions that prove the rule are L2 and L4, both of which areproteins belonging to the “glb+ext” class. Protein L2 was crystallizedand its structure solved only after its extensions had been removed(Nakagawa et al. (1999) supra), and one of the two regions of L4 thatare extended in the ribosome is disordered in the crystal structure ofintact L4 (Wahl et al. (2000) supra).

Except for proteins L1, L7, L10 and L11, which form the tips of the twolateral protuberances, the proteins of the 50S subunit do not extendsignificantly beyond the envelope defined by the RNA (see, FIG. 7).Their globular domains are found largely on the particle's exterior,often nestled in the gaps and crevices formed by the folding of the RNA.Thus, unlike the proteins in spherical viruses, the proteins of thelarge ribosomal subunit do not form a shell around the nucleic acid withwhich they associate, and unlike the proteins in nucleosomes, they donot become surrounded by nucleic acid either. Instead, the proteins actlike mortar filling the gaps and cracks between “RNA bricks.”

The distribution of proteins on the subunit surface is nearly uniform,except for the active site cleft and the flat surface that interactswith the 30S subunit. In the crown view the proteins lie around at theperiphery of the subunit (see, FIG. 7(A)), but when viewed from the sideopposite the 30S subunit binding site (the “back side”), they appear toform an almost uniform lattice over its entire surface (see, FIG. 7(B)).Similarly, the bottom surface of the subunit, which includes the exit ofpolypeptide tunnel, is studded with proteins (see, FIG. 7(C)). Indeed,the 5 proteins that surround the tunnel exit may play a role in proteinsecretion since they are part of the surface that faces the membrane andthe translocon when membrane and secreted proteins are beingsynthesized.

Although FIG. 7 shows protein chains disappearing into the ribosomeinterior, the degree to which proteins penetrate the body of theparticle can only be fully appreciated when the RNA is stripped away.The interior of the particle is not protein-free, but it is protein-poorcompared to the surface of the particle. Extended tentacles ofpolypeptide, many of which emanate from globular domains on the surface,penetrate into the interior, filling the gaps between neighboringelements of RNA secondary structure (see, FIG. 8(E)). The bizarrestructures of these extensions are explained by their interactions withRNA.

Although extended, non-globular structures are rare in the protein database, they are not unknown. Extended protein termini often forminter-protein contacts, e.g., in viral capsids, presumably adoptingfixed structures only upon capsid formation. The basic “tails” ofhistones may behave the same way when nucleosomes form. The N-terminalsequences of capsid proteins are often positively charged, and in viruscrystal structures, the electron density for these sequences oftendisappears into the interior of the virus where these sequencespresumably interact with asymmetrically arranged nucleic acid. Theinteractions observed in the ribosome could be useful models for theseviral interactions.

The interactions of extended polypeptides and RNA in the large subunit,which stabilizes its massive nucleic acid structure, result in a tangleof RNA and protein in the center of the subunit (see, FIGS. 8(A) and8(B)). It is hard to imagine such an object assembling from itscomponents efficiently in anything other than a highly ordered manner.Chaperones may well be required to prevent the aggregation of theextended regions of these proteins, which are likely to be disorderedoutside the context provided by rRNA, and to manage the folding of rRNA.

8. Protein and RNA Interactions.

Because protein permeates the large subunit to a surprising degree,there are only a few segments of the 23S rRNA that do not interact withprotein at all. Of the 2923 nucleotides in 23S rRNA, 1157 nucleotidesmake at least van der Waals contact with protein (see, FIG. 8(D)), andthere are only ten sequences longer than twenty nucleotides in which nonucleotide contacts protein. The longest such sequence containsforty-seven nucleotides, and is the part of domain IV that forms theridge of the active site cleft.

The extent of the interactions between RNA and protein that occur whenthe large subunit assembles can estimated quantitatively. Using theRichards algorithm (Lee, B. et al. (1971) J. Mol. Biol. 55: 379-400) anda 1.7 Å radius probe to compute accessible surface areas, it can beshown that 180,000 Å² of surface becomes buried when the subunit formsfrom its isolated, but fully structured components. This is about halftheir total surface area. The average is about 6,000 Å² per protein.While this is an enormous amount compared to the surface buried whenmost protein oligomers form, it should be recognized that ribosomeassembly must be accompanied by a large loss in conformational entropythat does not occur when most proteins oligomerize. The extended proteintermini and loops of the ribosomal proteins are almost certainlyflexible in isolation, and in the absence of protein, the RNA isprobably quite flexible as well. Thus, the burial of a large amount ofsurface area may be required to provide the energy necessary to fix ofthe structures of these molecules.

All of the proteins in the particle except L12, interact directly withRNA and all but seven of the remaining thirty proteins interact with tworRNA domains or more (Table 2). The “champion” in this regard is L22,which is the only protein that interacts with RNA sequences belonging toall 6 domains of the 23S rRNA (see, FIG. 8(C)). The protein-mediatedinteractions between 5S rRNA and 23S rRNA are particularly extensive.Protein L18 attaches helix 1 and helix ⅔ of 5S rRNA to helix 87 of 23SrRNA. Protein L31e mediates an interaction between the same part of 5SrRNA and domains II and V. Loop C is linked to domain V by protein L5and loop D is attached to domains II and V by protein L10e. Whateverelse they may do, it is evident that an important function of theseproteins is stabilization of the relative orientations of adjacent RNAdomains. Several also help secure the tertiary structures of the domainswith which they interact.

Since most ribosomal proteins interact with many RNA sequences and thenumber of proteins greatly exceeds the number of RNA domains, it canhardly come as a surprise that every rRNA domain interacts with multipleproteins (Table 2). Domain V, for example, interacts with thirteenproteins, some intimately and a few in passing.

It is clear that the oligonucleotide binding experiments long relied onfor information about the RNA binding properties of ribosomal proteinshave underestimated their potential for interacting with RNA. Thehigh-affinity RNA binding site identified on a protein by such anexperiment may indeed be important for ribosome assembly, but its many,weaker interactions with other sequences are likely to be missed, andthey too may be vital for ribosome structure. Most ribosomal proteinscrosslink RNA and crosslinking is impossible without multipleinteractions. Similar considerations may apply to proteins that arecomponents of other ribonucleoproteins such as the sliceosome.

Of the seven proteins that interact with only one domain, three (L1,L10, L11) participate directly in the protein synthesis process. Ratherthan being included in the ribosome to ensure that the RNA adopts theproper conformation, it seems more appropriate to view the RNA as beingstructured to ensure their correct placement. Another three (L24, L29,L18e) interact with several secondary structure elements within thedomains to which they bind, and presumably function to stabilize thetertiary structures of their domains. The last of the single RNA domainproteins, L7ae, is puzzling. On the one hand, it cannot function as anRNA stabilizing protein because it interacts with only a single, shortsequence in domain I, and on the other hand, it is far from theimportant functional sites in the subunit, the peptidyl transferase siteand factor binding site. It is quite close to L1, however, which appearsto be important for E-site function (Agrawal et al. (1999) J. Biol.Chem. 274: 8723-8729), and maybe it is involved in that activity.

While many ribosomal proteins interact primarily with RNA, a fewinteract significantly with other proteins. The most striking structuregenerated by protein-protein interactions is the protein clustercomposed of L3, L6, L14, L19 and L24e that is found close to the factorbinding site. The protein surface they provide may be important forfactor interactions.

The structure presented above illuminates both the strengths andweaknesses of approaches to complex assemblies that depend ondetermining the structures of components in isolation. The structures ofthe globular domains of homologues of the proteins in large ribosomalsubunit from H marismortui are largely the same as those of thecorresponding domains in the intact subunit, though adjustments indomain positions are sometimes required. Consequently, these structureswere very useful for locating proteins and interpreting lower resolutionelectron density maps. However, for obvious reasons, the structures ofthe extended tails and loops of ribosomal proteins cannot be determinedin the absence of the RNAs that give them structure, and the feasibilityof strategies that depend on producing low molecular weight RNA-proteincomplexes that have all the RNA contacts required to fix the structuresof such proteins seems remote. RNA is also a problem. While thesarcin/ricin loop has much the same structure in isolation as it does inthe ribosome, the structure of 5S rRNA in isolation differs in somerespects from what is seen in the ribosome, and the structure of theisolated P-loop (Puglisi et al. (1997) Nat. Struct. Biol. 4: 775-778)does not resemble the structure of the P-loop in the ribosome at all.Clearly a “structural genomics” approach to the ribosome, which wouldhave entailed determining the structures of all its proteins and allpossible rRNA fragments, would not have worked. It may not be successfulfor other macromolecular assemblies either.

B. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis

Analysis of the atomic co-ordinates discussed in section IIA abovetogether with additional atomic co-ordinates of a ribosomal subunitcomplexed with various analogues, similarly refined, permit an analysisof ribosome function. Accordingly, the present invention is also basedon the crystals of Haloarcula marismortui 50S ribosomal subunitcomplexed either with the Yarus transition state analogue, CCdA-p-Puro,or with a mini-helix analogue of an aminoacyl-tRNA. The presentinvention provides the structures of both complexes. The atomicco-ordinates of the structure of both complexes were deposited on Jul.26, 2000, at Research Collaboratory for Structural Bioinformatics (RCSB)Protein Data Bank (PDB) (Berman et al. (2000) Nucleic Acid Research 28:235-242; see also, the web page at the URL rcsb.org/pdb/) with accessionnumbers PDB ID: 1FFZ (50S ribosome/ CCdA-p-Puro complex) and PDB ID:1FG0(50S ribosome/aa-tRNA analogue).

As discussed below, the complete atomic structures of the largeribosomal subunit and its complexes with two substrate analogues showthat the ribosome is a ribozyme. The complete atomic structures alsoprovide information regarding the catalytic properties of its all-RNAactive site. Both substrate analogues are contacted exclusively byconserved rRNA residues from domain V of 23S rRNA; there are no proteinside-chains closer than about 18 Å to the peptide bond beingsynthesized. The mechanism of peptide bond synthesis appears to resemblethe reverse of the deacylation step in serine proteases, with the baseof A2486 (A2451) in E. coli playing the same general base role as His57in chymotrypsin. The unusual pKa that A2486 must possess to perform thisfunction probably derives from its hydrogen bonding to G2482 (G2447)which interacts with a buried phosphate that could stabilize the unusualtautomers of two bases which is required for catalysis. The polypeptideexit tunnel is largely formed by RNA but has significant contributionsfrom proteins L22, L39 and L4 and its exit is encircled by proteins L19,L22, L23a, L24 and L29.

The CCdA from the Yarus analogue binds to the so-called P-loop and hencemust be in the P-site. Only the terminal-CCA of the aa-tRNA analogue isvisible, but since it interacts appropriately with the A-loop (Kim etal. (1999) Molec. Cell 4: 859-864), it must be in the A-site. Thepuromycin group occupies the same location in both structures, and thereare no proteins near that site. Hence, the catalytic activity of theactive site must depend entirely on RNA. The N3 of A2486 (E. coli A2451)is the titratable group nearest to the peptide bond being synthesizedand is likely functioning as a general base to facilitate thenucleophilic attack by the a-amino group of the A-site substrate. Inorder to function in this capacity, the pKa of this base has to beroughly 5 units higher than normal.

1. Structures of Substrate Analogue Complexes.

In order to establish how substrates interact at the A-site and P-siteof the large subunit, two substrate analogues were used. One of theanalogues, which was designed to mimic the acceptor stem of an aa-tRNAand bind to the A-site, was a twelve base-pair RNA hairpin with anaminoacylated, four-nucleotide extension on its 3′ end (see, FIG. 9).The sequence used was that of the tRNA tyr acceptor stem and it isterminated with puromycin, which itself is an analogue of tyrosyl-A76.The second analogue used was the Yarus transition state analogue,CCdA-p-puromycin. As in the case of the A-site substrate analogue, thepuromycin of the Yarus inhibitor is expected to bind at the A-site,while its CCdA moiety should bind at the P-site.

The positions of the Yarus inhibitor and the tRNA acceptor stem analoguewere determined by soaking these molecules into crystals of the H.marismortui 50S ribosomal subunit, measuring diffraction data to 3.2 Åresolution and calculating difference electron density maps (Welch etal. (1997) Biochemistry 36: 6614-6623). Maps of the complexes were alsocalculated using 2F_(o)(complexed)- F_(o)(uncomplexed) as coefficients,to examine the shifts in the positions of ribosome residues that occurwhen these analogues bind (see, FIG. 10(B) and Table 3).

TABLE 3 Statistics for Data Collection and Scaling. Crystal Native ANative B CcdAp-Puro Mini-helix Soak time — —  24  24 (hours) Soak — —100 100 concentration (μM) Wavelength 1.0 1.0 1.0 1.0 (Å) Observations1,571,171 1,344,877 2,590,726 2,712,813 Unique 284,533 369,167 367,284447,121 Redundancy 5.5 3.6 7.0 6.0 Resolution 70.0-3.2 70.0-3.0 70.0-3.070.0-2.8 limits (Å) (High- (3.26-3.20) (3.05-3.00) (3.23-3.17)(3.08-3.02) resolution bin)* Completeness 94.1 (96.0) 98.9 (99.3) 98.6(99.9) 99.6 (100) l/σl 14.6 (4.0)  10.8 (3.1)  11.0 (2.8)  10.7 (2.9) R_(merge)† 10.2 (40)  11.5 (38)   18.8 (84)   14.3 (72)  R_(iso) NativeA‡ — —  6.8 (20.8)  14.4 (25.2) R_(iso) Native B‡ — — 12.6 (27.4)  17.5(31.0) *Statistics in parenthesis are calculated for the high-resolutionbin used in map calculations, which, as indicated was sometimes lower inresolution than the high-resolution bin used in data reduction.†R_(merge): ΣΣ₁|I_((h)) − I_((h)i)|IΣΣI_((h)i)′ where I_((h)) is themean intensity after reflection. ‡R_(iso): Σ|F_(PH) − F_(P)|ΣF_(PH) _(—)where F_(PH) and F_(P) are the soaked and the native crystal structurefactor amplitudes respectively.

A model for the entire Yarus inhibitor could be fitted into thedifference density (see, FIG. 10(A)), and the electron density map ofthe complex shows the N3 of A2486 (2451) within hydrogen bondingdistance of a non-bridging oxygen of the phosphoramide (see, FIG.10(B)). The inhibitor's two C's, which correspond to C74 and C75 ofpeptidyl-tRNA, are Watson-Crick base-paired with G2285 (2252) and G2284(2251) in the P-loop, respectively (see, FIG. 11(A)). The C74-G2285(2252) interaction was predicted by the results of Noller and coworkers(Noller et al. (1992) Science 256: 1416-1419). The dA, which correspondsto A76 of a tRNA in the P-site, is not base-paired, but rather stacks onthe ribose of A2486 and hydrogen bonds to the 2′OH of nucleotide A2485(see, FIG. 12(B)).

Only the CC-puromycin moiety of the mini-helix acceptor stem analogueshowed ordered electron density in its difference electron density map(see, FIG. 10(C)). The C75 of the acceptor stem CCA is Watson-Crickbase-paired with G2588 (2553) of the A-loop, whereas the C74 is moredisordered and not base-paired but appears to stack on a ribosome base.The dimethyl A of the A-site inhibitor puromycin is positionedidentically to the dimethyl A of the Yarus inhibitor. Further, thedimethyl A of puromycin, which is the A76 equivalent of an A-site tRNA,interacts with the A-loop in much the same way that the A76 from theP-site CCA interacts with the P-loop (see, FIG. 11(B)).

The most notable of the several conformational changes in the ribosomeinduced by the binding of the transition state analogue is the orderingof base A2637 (2602), which is disordered in the unliganded enzyme (see,FIG. 11(B)). It becomes positioned between the CCA bound at the A-siteand the CCA bound at the P-site. The base of U2620 (2585) also moves sothat it can make a hydrogen bond with the 2′ hydroxyl of the ribose ofA76 in the A-site, and U2619 and G2618 shift to allow the A76 to bepositioned. Smaller shifts in positions are observed in the positions ofA2486, whose N3 is near to the non-bridging oxygen of the phosphate, andone of the G residues with which it interacts, G2102 (2482).

2. Location and Chemical Composition of the Peptidyl Transferase Site.

The inhibitors are bound to a site made entirely of 23S rRNA with noproteins nearby, proving that the ribosome is a ribozyme. Both the Yarusinhibitor and the A-site analogue of aa-tRNA bind to the large subunitat the bottom of a large and deep cleft at the entrance to the 100 Ålong polypeptide exit tunnel that runs through to the back of thesubunit (see, FIG. 12). This site is surrounded by nucleotides belongingto the central loop of 23S RNA domain V, the “peptidyl transferaseloop.” Nucleotides from the single stranded portions of this loop makethe closest approach to the phosphate that mimics the tetrahedral carbonintermediate. In general, the helices that extend from the peptidyltransferase loop in 2 secondary structure diagrams of 23S rRNA alsoextend away from the active site in the tertiary structure (see, FIG.13). Although there are 13 proteins that interact with domain V (see,FIG. 14(A)), there are no globular proteins in the vicinity of theinhibitor. The closest polypeptides are the non-globular extensions ofseveral proteins (L2, L3, L4, L10e) that penetrate deeply into domain Vand approach the active site (see, FIG. 14(B)). These extensions fillmany of the voids between the RNA helices of domain V, neutralizephosphate backbone charge, and presumably stabilize the structure of thedomain and its association with other RNA regions. However, none oftheir side chain atoms is closer than about 18 Å to the phosphorus ofthe inhibitor's phosphate group, which marks the site where peptidebonds form. Furthermore, the substrate analogue is completely enclosedin an rRNA cavity that is so tightly packed that there is no possibilitythat an unidentified peptide could be lurking nearby (see, FIG. 15).Thus, the catalytic entity in the ribosome must be RNA.

Two of the proteins with long termini or loops penetrating the rRNAscaffold of domain V are proteins that could not previously be excludedfrom involvement in the peptidyl transferase reaction L2 and L3 (Noller(1991) Ann. Rev. Biochem. 60: 191-227). Noller and colleagues (Noller etal. (1992) supra) found that under conditions which prevent RNAdenaturation, extensive digestion of Thermus thermophilus 50S subunitswith proteases followed by extraction with phenol and other agents thatdisrupt protein-RNA interactions did not remove several peptides fromthe subunit that were less than 10,000 in molecular weight. Thestructure makes it clear why these protein fragments were particularlyresistant to protease treatments. While protease treatment could digestthe globular protein domains on the surface of the large subunit, itcould not remove the long termini or loops that penetrate deeply in the23S rRNA because they are sequestered within the rRNA and thus protectedfrom cleavage, independently of the globular domains.

3. Peptidyl Transferase Active Site.

The RNA that surrounds the substrate analogues is closely packed, muchlike the active site region of a protein enzyme and the nucleotides incontact with the inhibitor are greater than 95% conserved in all threekingdoms of life (see, FIG. 15). Thus, it is clear that the ribosome isa ribozyme, but what gives the RNA its catalytic power?

Without wishing to be bound by theory, the residue most likely to beinvolved in catalysis, presumably as a general base, is A2486, whose N3is about 3 Å from the phosphoramide oxygen of the Yarus inhibitor thatis the analogue of the carbonyl oxygen of a nascent peptide bond andabout 4 Å from the amide that corresponds to the amide nitrogen of thepeptide bond being synthesized. Ordinarily, the pKa of the N1 ofadenosine monophosphate is about 3.5 and that of its N3 is perhaps 2 pHunits lower (Saenger (1984) Principles of Nucleic Acid Structure, (C. R.Cantor, eds.), Springer Advanced Texts in Chemistry, Springer-Verlage,New York, N.Y.), and in order for A2486 to function as a general base,its pKa would have to be raised to 7 or higher. The crystal structureitself suggests that its pKa is, in fact, quite unusual. The N3 of A2486can only hydrogen bond to the phosphate oxygen, as observed, if it (orthe phosphate oxygen) is protonated. The distance between these twoatoms is about 3 Å indicating that a hydrogen bond does, indeed, existbetween them. Since the crystal is at pH 5.8, this implies that the pKaof the N3 is greater than 6. Muth and Strobel have measured the pKa ofthe corresponding A in E. coli 23S RNA by examining its dimethyl sulfatereactivity as a function of pH and have concluded that it is 7.6, thoughthey cannot be sure from their experiments whether it is the N3 or N1whose pKa they have measured (Muth et al. (2000) Science 289: 947-950).Because there is no other available, titratable RNA functional groupcloser than about 7 Å to the nascent peptide bond, there is not othergroup available to function as a general base.

There are several features of environment of A2486 (2451) that mightaffect its pKa. The pKa of the N3 of A2486 (2451) may be increasedsignificantly in part by a charge relay mechanism, analogous to thatwhich occurs in the active site of the serine proteases, with the buriedphosphate of A2485 (2450) performing a similar function as the buriedcarboxylate of Asp102 of chymotrypsin. The experimental 2.4 Å electrondensity map unambiguously establishes the hydrogen bonding interactionsin this most critical region of the active site (see, FIG. 16(A)). TheN6 of A2486 interacts with the 06 atoms of G2482 (2447) and G2102 (2061)(see, FIG. 16(B)). The N2 of G2482 is also interacting with anon-bridging oxygen of the phosphate group of A2485 (2450) that is amongthe total of 3 most solvent inaccessible phosphate groups (826, 1497 and2485) in the large ribosomal subunit for which we do not see anyneutralizing counterion in the 2.4 Å resolution map. Weak density thatmay correspond to a water molecule is hydrogen bonded to the othernon-bridging oxygen. A neutralizing counterion is not apparent in thisstructure. The buried phosphate of A2485 could abstract the proton fromthe exocyclic N2 of G2482 in order to neutralize its energeticallyunfavorable buried negative charge. This, in turn, would stabilize theotherwise rare imino tautomer of that base. The interaction of an iminoof G2482 with A2486 likewise can stabilize the imino tautomer of A2486that would result in a negative charge on its N3 were it unprotonated(see, FIG. 16(C)). In this way, some of the negative electrostaticcharge originating on the buried phosphate of A2485 could be relayed tothe N3 of A2486, thereby increasing its pKa.

A second feature of the environment of the catalytic site that mayaffect its stability, tautomeric state and electrostatic chargedistribution is a bound monovalent cation. A potassium or a sodium ioninteracts with the O6 of G2482 and G2102 as well as with three otherbases. Its identity as a potassium ion is established by its observedcontinuation and by an independent experiment showing that a rubidiumion can bind to this site. The monovalent ion might also stabilizenon-standard tautomers, but its expected influence on the pKa of A2486is less obvious. Early biochemical experiments have shown the importanceof potassium for peptidyl transferase activity (Monro (1967) supra;Maden et al. (1968) supra) and this binding site could be responsiblefor that affect.

It may also be the case that stabilization of an imino tautomer by aburied phosphate explains the expected higher pKa of a catalyticcytosine in the active site of the hepatitis delta virus ribozyme(Ferre-D'Amare et al. (1998) Nature 395: 567-574; Naharo et al. (2000)Science 287: 1493-1497). In this case, a backbone phosphate, whosesolvent accessibility is similar to that of A2485 in the ribosome, isobserved to hydrogen bond to the N4 of C, and the protonated form of theimino tautomer of that C would neutralize the phosphate, promoting thefunction of its N3 as a general acid (Naharo et al. (2000) supra).

4. Catalytic Mechanism of Peptide Bond Formation.

The proximity of the N3 of A2486 (2451) to the peptide bond beingsynthesized and the nature of the reaction catalyzed suggest a chemicalmechanism of peptide synthesis that is analogous to the reverse of thedeacylation step seen in serine proteases during peptide hydrolysis(Blow et al. (1969) Nature 221: 337-340; Steitz et al. (1982) Ann. Rev.Biophys. Bioeng. 11: 419-444). In that mechanism, the basic form ofHis57 abstracts a proton from the α-amino group of the peptide productas it attacks the acyl-Ser195. Formation of the tetrahedral carbonylcarbon intermediate is stabilized by interaction of the oxyanion formedwith backbone amides (the “oxyanion hole”); His57 shuttles the protonacquired from the α-NH₂ to Ser195 as the tetrahedral intermediate breaksdown.

The residue A2486 (2451) appears to be the analogous to His57 inchymotrypsin and that the peptidyl-tRNA is analogous to acyl-Ser195.Thus, the N3 of A2486, with its greatly elevated pKa, abstracts a protonfrom the a-amino group of the A-site bound aminoacyl-tRNA facilitatingthe nucleophilic attack of that amino group on the carbonyl carbon thatacylates the 3′ OH of the tRNA in the P-site (see, FIG. 17(A)). Incontrast to the serine proteases, however, the oxyanion of thetetrahedral intermediate is near to the protonated N3 of A2486 (A2451)rather than being proximal to a separate oxyanion binding site. Thus, itcould be that the protonated N3 of A2486 stabilizes the formation of theoxyanion by hydrogen bonding to it, as we observe in the Yarus inhibitorcomplex (see, FIG. 17(B)). The N3 of A2486 could then subsequentlytransfer its proton to the 3′ hydroxyl of the P-site bound tRNA, whichis liberated as the peptide shifts to the A-site bound tRNA (see, FIG.17(C)).

An additional question is how is the catalyzed hydrolysis of thepeptidyl tRNA in the P-site prevented prior to the delivery of the nextappropriate aa-tRNA to the A-site? It appears from this complex thatwater would not be excluded from access to the peptidyl link to theP-site tRNA if the A-site were vacant. An analogous problem wasdiscussed by Koshland in the 1960s (Koshland, Jr. (1963) Cold SpringHarbor Symp. Quant. Biol. 28: 473-489), who theorized why hexokinasedoes not hydrolyze ATP in the absence of glucose, since water shouldbind perfectly well to the binding site used by the 6-hydroxyl ofglucose. The answer proposed was induced fit, i.e., hexokinase is notcatalytically competent until the glucose binds and produces aconformational change that orients substrates and catalytic groupsoptimally. This indeed appears to be the case (Bennett, Jr. et al.(1978) Proc. Natl. Acad. Sci. USA 75: 4848-4852). Similarly, it could bethat the catalytic A2486 and/or the peptidyl substrate are not properlyoriented or that the binding site for the α-NH₂ group is blocked by areoriented ribosome base in the absence of aa-tRNA in the A-site. Thebase of U2620 appears close to A2486 in the ligand free structure, andit may serve as the appropriate plug that prevents spontaneoushydrolysis of peptidyl-tRNA.

Thus, it appears that this RNA enzyme uses the same principles ofcatalysis as a protein enzyme. First, a large catalytic enhancement isachieved by precisely orienting the two reactants, the αNH₂ from theA-site aminoacyl-tRNA and the carbonyl carbon from the P-sitepeptidyl-tRNA. This may be accomplished, in part, by the interactions ofthe CCA ends of the A-site and P-site tRNAs with the A-loop and P-loop,respectively. Secondly, acid-base catalysis and transition statestabilization are achieved by an enzyme functional group (A2486 (2451)in this case) whose chemical properties are altered appropriately by theactive site environment. Third, similar chemical principles may be usedby RNA and protein enzymes to alter the pKa's of functional groups. Aburied carboxylate of Asp102 acting through His57 alters thenucleophilicity of Ser195 in chymotrypsin (Blow et al. (1969) supra). Inthe ribosome a solvent inaccessible phosphate may act through G2482(2447) alters the nucleophilicity of the N3 of A2486 (2451). It could bethat RNA molecules “learned” how to use the chemical principles ofcatalysis significantly before protein molecules did.

5. tRNA Binding.

While it is not possible to bind tRNA molecules to either the A-orP-sites in these crystals for steric reasons, it is possible to placethe A-, P- and E-site tRNA molecules on the large ribosomal subunit inthe same relative orientation that Cate et al. observed in theircrystallographic study of the Thermus aquaticus 70S ribosome. Theco-ordinates of the three tRNA molecules in the relative positions seenin the 70S ribosome can be docked on the Haloarcula marismortui largeribosomal subunit in a way that avoids steric clashes and places theacceptor stems of the A-site and P-site tRNAs near to the positions ofthe CCAs we have observed bound to the A-loop and P-loop (see FIG. 18).Although nucleotides C74 and C75 were modeled in a differentconformation in the 7.8 Å ribosome map, the C74 residues from the CCAsin both the A- and P-sites can be connected to residue 72 of the dockedA-site and P-site tRNAs through a modeled residue 73, and it appearsthat the tRNA molecules fit well onto the surface of the subunit.Unexpectedly, this modeling places the E-site, P-site and A-site boundtRNA molecules in close proximity to three ribosomal proteins. ProteinsL5 and L10e are near tRNAs in the P-site and A-site. Since both of theseproteins also interact with 5S rRNA, this observation raises thepossibility that 5S rRNA and some of its associated proteins might helpstabilize the positioning of ribosome bound tRNAs and is consistent withthe fact that 5S rRNA enhances ribosomal activity, but is not absolutelyessential for it (Moore, Ribosomal RNA & Structure, Evolution,Processing and Function in Protein Biosynthesis (1996), supra, pp.199-236). Protein L44e appears to interact with the E-site tRNA and maycontribute to E-site activity. According to this docking experiment theA-site tRNA interacts with the highly conserved stem-loop 2502-2518(2467-2483) which together with L10e forms a large concave surface thatcontacts the tRNA on the T-stem, utilizing the exact same binding siteexploited by EF-Tu (Gutell et al. (2000) supra).

Examination of the relationships between the CCAs bound in the A- andP-sites and the tRNAs to which they are connected as well as theirinteractions with the ribosome also leads to some insights intotranslocation. Immediately after formation of the new peptide bond anddeacylation of the P-site tRNA, the acceptor end of the P-site tRNA isknown to move to the E-side and that of the A-site tRNA moves to theP-site (Blobel et al. (1970) J. Cell. Biol. 45: 130-145). Theapproximate modeling of the 3 tRNA molecules on the large subunitsuggests some possible contributions to this process. First, there aretwo base-pairs between the P-site tRNA and the P-loop and only onebetween the A-site and the A-loop. Moving from the A- to the P-siteincreases base-pairing, though there must be a concomitant attraction ofthe deacylated P-site tRNA to an E-site. Further, the CCAs bound to theA and P loops are related by 180° rotation, whereas the tRNAs to whichthey are attached are not. Thus, the relationships of the CCAs to theacceptor stems cannot be the same in both sites and may not be equallystable. If the conformation of the A-site tRNA is less stable, thenmoving a tRNA from the A- to the P-site might be energetically favored.

6. Polypeptide Exit Tunnel.

It appears very likely from the structure that all nascent polypeptidespass through the polypeptide exit tunnel before emerging from theribosome, because there appears to be no other way out. We are now ableto address two important questions about the functioning of thepolypeptide exit tunnel: (1) Why do nascent proteins not stick to itswalls? Teflon has the marvelous property of not sticking to denaturedegg proteins, so how has the ribosome achieved a similar non-sticksurface for the denatured proteins that must pass through the tunnel?(2) Do proteins fold to any degree in the tunnel giving the ribosome achaperon-like function? The length of the tunnel from the site ofpeptide synthesis to its exit is about 100 Å, broadly consistent withthe length of nascent polypeptide that is protected from proteolyticcleavage by the ribosome (Moazed et al. (1989) Nature 342:142) and theminimum length required for antibody recognition at the exit (Picking etal. (1992) Biochemistry 31: 2368-2375). The tunnel is largely straight,except for a bend 20 to 35 Å from the peptidyl transferase center (see,FIG. 19). Its diameter varies from about 20 Å at its widest to a narrowpoint of about 10 Å at the very beginning and at a position 28 Å fromthe tunnel exit with an average diameter of about 15 Å. Since thesmallest orifice through which the polypeptide product must pass onlybarely accommodates the diameter of an a-helix diameter, it seemsunlikely that significant protein folding beyond the formation ofa-helix could occur within the ribosome.

The majority of the tunnel surface is formed by domains I-V of 23S rRNA,but significant contributions are also made by the non-globular regionsof proteins L22, L4 and L39 which not only fill some of the voids in theRNA scaffold, but also form significant portions of the tunnel wall(see, FIG. 19). The largest protein contributor to the surface of thetunnel is L22 whose long a-hairpin loop lies between RNA segments ofdomains I through IV and is approximately parallel with the axis of thetunnel. Unlike the other tunnel proteins, protein L39 does not have aglobular domain at the surface of the particle and is almost entirelyburied in domains I and III underneath protein L23. Interestingly, thenucleotides of 23S rRNA that form the tunnel wall are predominantly fromloops in the 23S rRNA secondary structure (see, FIG. 19). As itprogresses through the tunnel from the active site, a nascentpolypeptide first encounters domain V followed 20 Å further along bydomains II and IV and proteins L4 and L22. The last half of the tunnelis formed by domains I and III and the protein L39e.

The narrowest part of the tunnel is formed by proteins L22 and L4 whichapproach the tunnel from opposite sides forming what appears to be agated opening (see, FIG. 19C). The function of this constriction, ifany, is not obvious. It might be the place where the nature of thenascent chain is sensed and the information transmitted to the surfaceof the particle, perhaps through L22 or L4. The a-hairpin of L22 at thesite of this orifice and the 23S rRNA interacting with it are highlyconserved; its globular portion is located adjacent to the tunnel exiton the surface that must face the translocon during protein secretion(see, FIG. 19).

The “non-stick” character of the tunnel wall must reflect a lack ofstructural and polarity complementarity to any protein sequence orconformation that it encounters. The tunnel surface is largelyhydrophilic and includes exposed hydrogen bonding groups from bases,backbone phosphates and polar protein side-chains (see, FIG. 19). Whilethere are many hydrophobic groups (sugars, bases, protein side-chains)facing the tunnel as well, there are no patches of hydrophobic surfacelarge enough to form a significant binding site for hydrophobicsequences in the nascent polypeptide. As the tunnel is some 20 Å indiameter and filled with water and the newly synthesized polypeptide ispresumably freely mobile, the binding of a peptide to the tunnel wallwould result in a large loss of entropy that would have to becompensated for by a large complementary interaction surface that islarger than 700 Å (Chothia et al. (1975) Nature 256: 705-708).Similarly, while Arg and Lys side-chains from a nascent peptide mayindeed interact with the phosphates exposed in the tunnel, the degree ofstructural complementarity and the net binding energy obtained afterdisplacing bound counterions must be too small to overcome the largeunfavorable entropy of immobilization that would result from peptidebinding. Thus, although the ribosome tunnel is made primarily of RNA,the nature of its surface is reminiscent of the interior surface of thechaperonin, GroEL (Xu et al. (1998) J. Struct. Biol. 124:129-141) in itsnon-binding conformation. Only in the conformation that exposes a largehydrophobic surface does GroEL bind a denatured protein.

There are six proteins (L19, L22, L23, L24, L29 and L31e) located at theexit from the tunnel, facing the translocon onto which the ribosomedocks during protein secretion. There is evidence that the ribosomebinds the translocon even after extensive digestion of its protein byprotease implying that interaction between the translocon and theribosome is mediated by RNA. The proximity of these proteins to thetranslocon, however, leads us to wonder what role, if any, they mightplay in the protein secretion process. Recent data from the Dobbersteinlaboratory shows that the N-terminal domain of SRP54, the G-protein fromthe signal recognition particle involved in signal peptide binding, canbe crosslinked to ribosomal proteins L23 and L29. These two proteins areadjacent to each other and at the tunnel exit (see, FIG. 19).

7. Evolution.

In vitro evolution of RNA oligonucleotides has produced small RNAmolecules that can bind molecules like the Yarus inhibitor effectivelyor catalyze the peptidyl transfer reaction (Zhaug et al. (1998) Chem.Biol. 5: 539-553; Welch et al. (1997) supra). The sequence and secondarystructure of one of these selected RNAs is reminiscent of the peptidyltransferase loop in domain V of 23S rRNA (Zhaug et al. (1998) supra).The most striking similarity is a five nucleotide sequence that isidentical to a sequence in domain V that includes the catalytic A2486,G2482 and the buried phosphate of A2485. Remarkably, all of the groupsinvolved in the proposed charge relay system for activating A2486 in theribosome, are present in the in vitro selected ribozyme. Thus, thoughthe surrounding structural context is likely to be different, it seemsplausible that this artificially evolved ribozyme uses the samemechanisms as the ribosome for shifting the pKa of an adenine andlikewise uses it as a base for peptide synthesis. A second RNA (Welch etal. (1997) supra) contains a 12 nucleotide loop that includes a 9-basesequence identical to that found in the same region of the peptidyltransferase loop.

The striking similarities between the sequences containing the keycatalytic elements found in the peptidyl-transferase active site of theribosome and sequences of in vitro selected RNAs having relatedactivities make it clear that the appearance of a small RNA domaincapable of catalyzing peptidyl transferase was a plausible first step inthe evolution of protein synthesis on the ribosome. The first peptidessynthesized by this primordial peptide synthesizing enzyme might havebeen random polymers or copolymers, and it may have functioned withsubstrates as simple as an aminoacylated CCA. Basic peptides of thetypes observed to form the non-globular extensions that co-fold with the23S rRNA might have been among the first peptides synthesized that werefunctionally useful. Such peptides may have enhanced the stability ofthe protoribosome and other early ribozymes as the more sophisticatedpeptides of the present day ribosome appear to do.

C. Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution,Complete Refinement

The three-dimensional structure of the large ribosomal subunit fromHaloarcula marismortui has now been completely refined at 2.4 Åresolution. The model includes 2876 RNA nucleotides, 3701 amino acidsfrom 28 ribosomal proteins, 117 magnesium ions, 88 monovalent cations,and 7898 water molecules. Many of its proteins consist of asurface-exposed globular domain and one or more basic, non-globularextensions that are buried in rRNA. Half of them include motifs commonin non-ribosomal proteins including, for example: RRM domains, SH3-likebarrels and zinc fingers. Proteins that have significant sequence andstructural similarity, such as L15 and L18e, make essentially identicalinteractions with rRNA.

More particularly, the H. marismortui 50S subunit has been completelyrebuilt and refined by successive rounds of gradient energy minimizationand B-factor refinement using CNS (Brünger et al. (1998) supra).Ribosomal proteins and rRNA were completely rebuilt using the softwareprogram “O” (Jones, T. A. et al. (1991) Acta Crystallogr. A46: 110-119)with 2F_(o)-Fe electron density maps prior to the modeling of solventand metal ions. Modeling errors in the proteins were identified usingPROCHECK (Laskowski et al.(1993) J. Appl. Cryst. 26: 283-291) and byinspection of F_(o)-Fc maps. Difference maps also aided in theidentification of errors in the rRNA, most often associated with sugarpuckers. In the process, some adjustments were made in amino acidconformations, sequence register and in sequences themselves. Sequencechanges made were largely limited to L10e, L15e, and L37Ae, the onlythree proteins from the H. marismortui 50S that have not been sequenceddirectly. In addition, fifty-one amino acids were added to the modeldescribed in section IIA with forty-four of these coming from L10 at thebase of the L7/L12 stalk and L39e which lines a portion of the wall ofthe polypeptide exit tunnel. Fewer adjustments were made to the rRNAstructure. Forty-nine new nucleotides were modeled and refined, mainlyin helices 43 and 44 in domain II of 23S rRNA. In addition, the sugarpucker or conformation about the glycosidic bond was adjusted for somenucleotides. The refinement process was monitored by the quality ofelectron density maps calculated using phases derived from the model aswell as R/R_(free) values. The completely refined model now includes2876 RNA nucleotides, 3701 amino acids, 210 metal ions, and 7898 watermolecules. The model refines to an R/R_(free) of 18.9%/22.3% and hasexcellent geometry (Table 4).

Solvent modeling began with the generation of a list of possiblemagnesium ions obtained by an automatic peak selection using CNS. Peaksgreater than 3.5σ in F_(o)-Fc maps positioned within the magnesiuminner-sphere coordination distance of 1.9-2.1 Å from N or O atoms wereselected. The resulting list was manually inspected and only peaks thatdisplayed clear octahcdral coordination geometry were selected asmagnesium.

Monovalent cations were identified on the basis of isomorphousdifferences between rubidium-soaked and native crystals of the H.marismortui 50S subunit. Since native crystals were stabilized in thepresence of 1.6M NaCl, these sites were initially modeled and refined asNa⁺¹. Refinement of these sites as K⁺¹ almost always resulted inunusually high temperature factors, with two exceptions where we havemodeled K⁺¹. Most of the monovalent sites in the 50S subunit appear tooccupied by Na⁺¹ in our crystals, however, these sites are likely to beoccupied by K⁺¹ in vivo.

Waters were selected as peaks greater than 3.5 σ in F_(o)-Fc electrondensity maps and between 2.5 and 3.3 Å of O or N atoms. IndividualB-factor values were used to assess the assignment of water molecules. Anumber of waters refined to B-factors significantly lower thansurrounding RNA and protein atoms. In many cases these peaks were foundto be metal or chloride ions. A small number of low B-factor watermolecules were retained in the final model because they could not beunambiguously assigned as other species. As a result of adding metalions and water molecules the final model now contains 98, 547non-hydrogen atoms. The refinement and model statistics for the largeribosomal subunit are summarized in Table 4.

TABLE 4 Refinement and Model Statistics for the H. marismortui 50SSubunit Space Group C222₁ a = 211.66 Å, b = 299.67 Å, c = 573.77 Å Totalnon-hydrogen atoms 98,542 RNA atoms 61,617 Protein atoms 28,800 Watermolecules 7,893 Magnesium ions 117 Potassium ions 2 Sodium ions 86Chloride ions 22 Cadmium ions 5 Refinement Statistics: Resolution Range15.0-2.4 Å Number of reflections used in refinement 623,525 Number ofreflections for cross-validation 6,187 R_(working) 18.9% R_(free) 22.3%σ_(a) coordinate error (cross-validated) 0.35 Å (0.43 Å) luzzaticoordinate error (cross-validated) 0.29 Å (0.35 Å) Deviations fromideality: r.m.s.d. bond lengths 0.0052 Å r.m.s.d. bond angles 1.13°r.m.s.d. dihedrals 15.7° r.m.s.d. impropers 2.12° Protein Statisticsfrom Ramachandran Plot: Residues in most favored regions 2704 (86.6%)Residues in additional allowed regions 379 (12.1%) Residues ingenerously allowed regions 27 (0.9%) Residues in disallowed regions 13(0.4%) Average B-factor Statistics (Å²): All atoms (high/low) 44.3(10.1/133.7) rRNA 41.2 (11.78/125.0) proteins 49.7 (13.9/92.5) waters41.89 (9.58/115.4)

Refinement has also permitted additional modeling of L10, L39e, and theL11 binding site in 23S rRNA. Furthermore, it has been discovered thatcertain motifs, for example, RRM topologies, SH3-like barrels and zincfingers are common in the 50S proteins and each recognizes rRNA in manydifferent ways. Proteins that have significant three-dimensionalhomology, however, such as L15 and L18e as well as L18 and S11, makeessentially identical interactions with rRNA. Additional structuralhomologies between 50S proteins and non-ribosomal proteins also areapparent. The solvent exposed surfaces of these globular protein domainsare rich in aspartate and glutamate residues, while irregular proteinextensions penetrate the RNA core of the ribosome. These extensions areoften highly conserved, and their abundance of arginine, lysine, andglycine residues is important for their function. Collectively, theresults show evolutionary connections between many ribosomal proteinsand illustrate that protein-RNA interactions in the ribosome, althoughlargely idiosyncratic, share some common principles.

D. Antibiotic Binding Sites

In addition to the foregoing structural studies, the structure of thelarge ribosomal subunit of H. marismortui has been determined complexedwith each of nine different antibiotics. More specifically, crystals ofthe H marismortui large ribosomal subunit have been soaked with one ofthe following antibiotics: anisomycin, blasticidin, carbomycin A,tylosin, sparsomycin, virginiamycin M, spiramycin, azithromycin,linezolid or erythromycin. The structure of the large ribosome subunitcomplexed with each antibiotic was then resolved based on X-raydiffraction data generated for each crystal.

Briefly, a small amount of a concentrated antibiotic solution was addedto a large subunit crystal suspended in stabilization solution andincubated for several hours. Following freezing and the other proceduresnormally used to prepare such crystals for experimental use, X-raydiffraction data were collected from the antibiotic containing crystals.Because the crystals were isomorphous to those from which the structuredescribed above was derived, the phases obtained for native crystal werecombined with the diffraction intensities obtained from theantibiotic-soaked crystal to obtain a structure for the latter. Theposition of the antibiotic in the crystal to which was bound is revealedmost clearly in difference electron density maps, which are electrondensity maps computed using the phases just referred to and amplitudesobtained by subtracting the amplitudes of crystals that contain noantibiotic from the (suitably scaled) amplitudes of those that containantibiotic. By using the foregoing methods, it was possible to determinethe atomic co-ordinates that show the spatial relationship betweenparticular antibiotics and their binding sites within the largeribosomal subunit. It is contemplated that similar methods can be usedto resolve the structure of other antibiotics complexed to the largeribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withanisomycin are listed in a table on compact disk, Disk No. 1 under thefile name anisomycin.pdb, a more refined set of which is represented ina table on compact disk, Disk No. 1 under the file name ANISOMYC.PDB,and deposited at the RCSB Protein Data Bank with the accession numberPDB ID: 1K73. In addition, FIG. 20 shows the spatial relationshipbetween the antibiotic anisomycin and the large ribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withblasticidin are listed in a table on compact Disk No. 1 under the filename blasticidin.pdb, a more refined set of which is represented in atable on compact disk, Disk No. 1 under the file name BLASTICI.PDB, anddeposited at the RCSB Protein Data Bank with the accession number PDBID: 1KC8. FIG. 21 shows the spatial relationship between the antibioticblasticidin and the large ribosomal subunit. For orientation, FIG. 21also includes a substrate for the P-site.

The atomic co-ordinates of the large ribosomal subunit complexed withcarbomycin A are listed in a table on compact disk, Disk No. 1 under thefile name carbomycin.pdb, a more refined set of which is represented ina table on compact disk, Disk No. 1 under the file name CARBOMYC.PDB,and deposited at the RCSB Protein Data Bank with the accession numberPDB ID: 1K8A. FIG. 22 shows the spatial relationship between theantibiotic carbomycin A and the large ribosomal subunit. FIG. 22 alsoshows a portion of the polypeptide exit tunnel.

The atomic co-ordinates of the large ribosomal subunit complexed withtylosin are listed in a table on compact disk, Disk No. 1 under the filename tylosin.pdb, a more refined set of which is represented in a tableon compact disk, Disk No. 1 under the file name TYLOSIN.PDB, anddeposited at the RCSB Protein Data Bank with the accession number PDBID: 1K9M. FIG. 22 shows the spatial relationship between the antibiotictylosin and the large ribosomal subunit. FIG. 22 also shows a portion ofthe polypeptide exit tunnel.

The atomic co-ordinates of the large ribosomal subunit complexed withsparsomycin are listed in a table on compact disk, Disk No. 1 under thefile name sparsomycin.pdb, a more refined set of which is represented ina table on compact disk, Disk No. 1 under the file name SPARSOMY.PDB.FIG. 23 shows the spatial relationship between the antibioticsparsomycin and the large ribosomal subunit. For orientation, FIG. 23also shows a substrate for the P-site.

The atomic co-ordinates of the large ribosomal subunit complexed withvirginiamycin M are listed in a table on compact disk, Disk No. 1 underthe file name virginiamycin.pdb, a more refined set of which isrepresented in a table on compact disk, Disk No. 1 under the file nameVIRGLNIA.PDB. FIG. 24 shows the spatial relationship between theantibiotics virginiamycin M as well as carbomycin A, and the largeribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withspiramycin are listed in a table on compact disk, Disk No. 1 under thefile name spiramycin.pdb, a more refined set of which is represented ina table on compact disk, Disk No. 1 under the file name SPIRAMYC.PDB,and deposited at the RCSB Protein Data Bank with the accession numberPDB ID: 1KD1. FIG. 25 shows the spatial relationship between theantibiotic spiramycin and the large ribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withazithromycin are listed in a table on compact disk, Disk No. 1 under thefile name AZITHROM.PDB, a more refined set of which is represented in atable on compact disk, Disk 1 under the file name azithromycin.pdb. FIG.26 shows the spatial relationship between the antibiotic azithromycinand the large ribosomal subunit.

The atomic co-ordinates of the large ribosomal subunit complexed withlinezolid are listed in a table on compact disk, Disk No. 1 under thefile name LINEZOLI.PDB, a more refined set of which is represented in atable on compact disk, Disk No. 1 under the file name linezolid.pdb.FIG. 27 shows the spatial relationship between the antibiotic linezolidand the large ribosomal subunit.

The atomic coordinates of the large ribosomal subunit complexed witherythromycin are listed in a table on compact disk, Disk No. 1 under thefile name erythromycin.pdb. FIG. 28 shows the spatial relationshipbetween the antibiotic erythromycin and the large ribosomal subunit.

FIG. 29 shows the spatial orientations of several antibiotics, namely,blasticidin, anisomycin, virginiamycin M and carbomycin A, as they bindto their respective antibiotic binding sites within the large ribosomalsubunit. For purposes of orienting the reader, the positions of theP-site, A-site and the polypeptide exit tunnel are shown in FIG. 29. Asis apparent, these antibiotics bind to or contact specific locationswithin the large ribosomal subunit to disrupt protein biosynthesis. Forexample, it appears that blasticidin binds the large ribosomal subunitin the vicinity of the P-site; anisomycin and virginiamycin bind thelarge ribosomal subunit in the vicinity of the A-site; and carbomycinspiramycin, tylosin, azithromycin and erythromycin (macrolide 5) allbinds the large ribosomal subunit in the vicinity of the polypeptideexit tunnel adjacent the peptidyl transferase site.

From FIG. 29, it is apparent that the skilled artisan may identifycertain portions of each antibiotic that contact regions in the largeribosomal subunit. By knowing their spatial relationship with respectone another, the skilled artisan may generate a hybrid antibioticmolecule comprising a portion of a first template antibiotic and aportion of a second, different template antibiotic. The two portions maybe linked by a chemical linker so as to maintain the spatial orientationof one portion with respect to the other portion. As a result, thehybrid antibiotic may simultaneously bind each of the regions of theribosomal subunit typically bound by each template antibiotic. Thedesign and testing of such molecules is discussed in more detail below.

FIG. 30(A) shows the tylosin molecule bound within the polypeptide exittunnel. FIG. 30(A) shows an enlarged portion of the large ribosomalsubunit with the antibiotic tylosin bound at the top of the polypeptideexit tunnel adjacent the peptidyl transferase site. FIGS. 30(B) and30(C) are views showing each half of a large ribosomal subunit cut alongthe polypeptide exit tunnel and are provided to orient the reader toshow the tylosin binding site relative to the large ribosomal unit as awhole. FIG. 30(A) also shows two cavities defined by the wall of thepolypeptide exit tunnel and are denoted as “cavity 1” and “cavity 2.” Inaddition, FIG. 30(A) also shows a disaccharide binding pocket. Thedirection in which the newly synthesized polypeptide chains exits theribosome through the polypeptide exit tunnel is denoted by an arrow.

E. Experimental Techniques Which Exploit X-Ray Diffraction Data

Based on the X-ray diffraction pattern obtained from the assemblage ofthe molecules or atoms in a crystalline solid, the electron density ofthat solid may be reconstructed using tools well known to those skilledin the art of crystallography and X-ray diffraction techniques.Additional phase information extracted either from the diffraction dataand available in the published literature and/or from supplementingexperiments may then be used to complete the reconstruction.

For basic concepts and procedures of collecting, analyzing, andutilizing X-ray diffraction data for the construction of electrondensities see, for example, Campbell et al. (1984) BiologicalSpectroscopy, The Benjamin/Cummings Publishing Co., Inc., (Menlo Park,Calif.); Cantor et al. (1980) Biophysical Chemistry, Part II: Techniquesfor the study of biological structure and function, W. H. Freeman andCo., San Francisco, Calif.; A. T. Brünger (1993) X-PLOR Version 3.1: Asystem for X-ray crystallography and NMR, Yale Univ. Pr., (New Haven,CT); M. M. Woolfson (1997) An Introduction to X-ray Crystallography,Cambridge Univ. Pr., (Cambridge, UK); J. Drenth (1999) Principles ofProtein X-ray Crystallography (Springer Advanced Texts in Chemistry),Springer Verlag; Berlin; Tsirelson et al. (1996) Electron Density andBonding in Crystals: Principles, Theory and X-ray DiffractionExperiments in Solid State Physics and Chemistry, Inst. of Physics Pub.;U.S. Pat. Nos. 5,942,428; 6,037,117; 5,200,910 and 5,365,456 (“Methodfor Modeling the Electron Density of a Crystal”).

A molecular model may then be progressively built using the experimentalelectron density information and further refined against the X-raydiffraction data resulting in an accurate molecular structure of thesolid.

F. Structural Determination Of Other Large Ribosomal Subunits

It is understood that the skilled artisan, when provided with the atomicco-ordinates of a first macromolecule may use this information toquickly and easily determine the three-dimensional structure of adifferent but structurally related macromolecule. For example, theatomic co-ordinates defining the large ribosomal subunit from H.marismortui can be used to determine the structure of the largeribosomal subunit from other species either as an isolated subunit, incomplex with the small subunit, or either of these complexed withfunctionally important ligands, for example: aminoacyl tRNA; variousprotein synthesis factors, such as elongation factor G, elongationfactor Tu, termination factor or recycling factor, in both their GTP andGDP conformational states; and protein synthesis inhibitors, forexample, antibiotics. In addition, the H marismortui subunitco-ordinates can also be used to solve the structures of ribosomalcomplexes with components of the protein secretion machinery, forexample, the signal recognition particle, and the translocon.

If the crystal being examined contains a macromolecule of unknownstructure and no additional information is available, additionalexperiments sometimes may be required to determine the relevant phasesof the macromolecule. These studies can often be time consuming anduncertain of success (Blundell et al. (1976) supra). However, whenadditional information, for example, structural and/or crystallographicinformation, is available for molecules related in some way to themacromolecule of interest then the process of resolving the structure ofthe molecule of interest is a much less challenging and time-consumingtask.

Accordingly, the skilled artisan may use information gleaned from theprior resolved structure to develop a three-dimensional model of a newmolecule of interest. Furthermore, the skilled artisan may use a varietyof approaches to elucidate the three-dimensional structure of the newmolecule. The approaches may depend on whether crystals of the moleculeof interest are available and/or whether the molecule of interest has ahomologue whose structure has already been determined.

In one approach, if the molecule of interest forms crystals that areisomorphous, i.e., that have the same unit cell dimensions and spacegroup as a related molecule whose structure has been determined, thenthe phases and/or co-ordinates for the related molecule can be combineddirectly with newly observed amplitudes to obtain electron density mapsand, consequently, atomic co-ordinates of the molecule of interest. Theresulting maps and/or atomic co-ordinates may then be refined usingstandard refinement techniques known in the art. In another approach, ifthe molecule of interest is related to another molecule of knownthree-dimensional structure, but crystallizes in a different unit cellwith different symmetry, the skilled artisan may use a technique knownas molecular replacement to obtain useful phases from the co-ordinatesof the molecule whose structure is known (Blundell et al. (1976) supra).This approach reportedly was used in the determination of the structureof the 50S subunit of Deinococcus radiodurans (Harms J. et al., Cell107(5):679-88; Schlunzen F. et al., (2001) Nature 413(6858):814-21)phases can then be used to generate an electron density map and/oratomic co-ordinates for the molecule of interest. In another approach,if no crystals are available for the molecule of interest but it ishomologous to another molecule whose three-dimensional structure isknown, the skilled artisan may use a process known as homology modelingto produce a three-dimensional model of the molecule of interest. It iscontemplated that other approaches may be useful in deriving athree-dimensional model of a molecule of interest. Accordingly,information concerning the crystals and/or atomic co-ordinates of onemolecule can greatly facilitate the determination of the structures ofrelated molecules.

The method of molecular replacement, developed initially by Rossmann andBlow in the 1960s, is now used routinely to establish the crystalstructures of macromolecules of unknown structures using the structureof a homologous molecule, or one in a different state of ligation (M. G.Rossmann, ed. “The Molecular Replacement Methods,” Int. Sci. Rev. J No.13, Gordon & Breach, New York, N.Y. (1972); Eaton Lattman, “Use ofRotation and Translation Functions,” H. W. Wyckoff C. H. W. Hist. (S. N.Timasheff, ed.) Methods in Enzymology, 115: 55-77 (1985)). For anexample of the application of molecular replacement, see, for example,Rice, P. A. & Steitz, T. A. (1994) EMBO J. 13: 1514-24.

In molecular replacement, the three-dimensional structure of the knownmolecule is positioned within the unit cell of the new crystal byfinding the orientation and position that provides the best agreementbetween observed diffraction amplitudes and those calculated from theco-ordinates of the positioned subunit. From this modeling, approximatephases for the unknown crystal can be derived. In order to position aknown structure in the unit cells of an unknown, but related structure,three rotation angles and three translations relative to the unit cellorigin have to be determined. The rotation search is carried out bylooking for agreement between the Patterson function of the search andtarget structures as a function of their relative orientation (therotation function). X-PLOR (Brünger et al. (1987) Science 235:458-460;CNS (Crystallography & NMR System, Brünger et al., (1998) Acta Cryst.Sect. D 54: 905-921), and AMORE: an Automatic Package for MolecularReplacement (Navaza, J. (1994) Acta Cryst. Sect. A, 50: 157-163) arecomputer programs that can execute rotation and translation functionsearches. Once the orientation of a test molecule is known, the positionof the molecule must be found using a translational search. Once theknown structure has been positioned in the unit cell of the unknownmolecules, phases for the observed diffraction data can be calculatedfrom the atomic co-ordinates of the structurally related atoms of theknown molecules. By using the calculated phases and X-ray diffractiondata for the unknown molecule, the skilled artisan can generate anelectron density map and/or atomic co-ordinates of the molecule ofinterest.

By way of example, it is contemplated that a three-dimensional model ofa ribosomal subunit other than that derived from H. marismortui can begenerated via molecular replacement. In this method, the H. marismortuisubunit structures are positioned within the unit cell of the newcrystal by finding the orientation and position that provides the bestagreement between observed diffraction amplitudes and those calculatedfrom the co-ordinates of the positioned subunit. A starting electrondensity map calculated using 2F_(hkl)(observed)−F_(hkl)(calculated),where F(observed) are the diffraction amplitudes that have been measuredfrom crystals of the unknown structure, and F(calculated) are thediffraction amplitudes calculated from the positioned H. marismortuisubunit structure. Refinement of the initial model can be done as isstandard in the field of macromolecular crystallography.

The H. marismortui 50S structure can also be used to establish thestructure of a 70S ribosome or 50S ribosome for which an electiondensity map has been calculated, at a resolution that would otherwise betoo low to be interpreted, while a 5 Å resolution map could not beinterpreted in atomic terms de novo, a plausible atomic model can beconstructed by refitting the H. marismortui 50S structure to a lowerresolution map (e.g., 4.5 Å to 8 Å). This refitting can be combined withhomology modeling to obtain a three-dimensional model of a ribosome orribosomal subunit from a different species. It is contemplated thatsimilar procedures may be used to determine the structure of theeukaryotic 60S subunit and/or a eukaryotic ribosome.

In general, the success of molecular replacement for solving structuresdepends on the fraction of the structures that are related and theirdegree of identity. For example, if about 50% or more of the structureshows an r.m.s. difference between corresponding atoms in the range ofabout 2 Å or less, the known structure can be successfully used to solvethe unknown structure.

Homology modeling, also known as comparative modeling or knowledge-basedmodeling, can be used to generate a three-dimensional model for amolecule based on the known structure of homologues. In general, theprocedure may comprise one or more of the following steps: aligning theamino acid or nucleic acid sequence of an unknown molecule against theamino acid or nucleic acid sequence of a molecule whose structure haspreviously been determined; identifying structurally conserved andstructurally variable regions; generating atomic co-ordinates for core(structurally conserved) residues of the unknown structure from those ofthe known structure(s); generating conformations for the other(structurally variable) residues in the unknown structure; building sidechain conformations; and refining and/or evaluating the unknownstructure.

By way of example, since the nucleotide sequences of all known 50Ssubunit rRNAs can be aligned relative to each other and to H.marismortui 23S and 5S rRNAs, it is possible to construct models of thestructures of other 50S ribosomal rRNAs, particularly in the regions ofthe tunnel and active sites, using the H. marismortui structure.Likewise, homologous proteins can also be modeled using similarmethodologies. Methods useful for comparative RNA sequence analysis areknown in the art and include visual methods and number pattern methods,as well as methods employing chi-square statistics, phylogeneticalgorithms, or empirical algorithms. Descriptions of some of theforegoing methods are available, for example, at on the world wide webat the URL ma.icmb.utexas.edu/; Gutell (1996), “Comparative SequenceAnalysis and the Structure of 16S and 23S rRNA,” Ribosomal RNA.Structure, Evolution, Processing, and Function in Protein Biosynthesis,(Dahlberg A. and Zimmerman B., eds.) CRC Press. Boca Raton, pp. 111-128;Guttell et al. (1993) Nuci. Acid Res. 21 :3055-3074; Schnare et al.(1996) J. Mol. Biol. 256: 701-719. Particularly useful visual inspectionmethods include comparison of a particular position in a H. marismortuisecondary structure diagram with the residues located at the analogousposition on an E. coli secondary structure diagram. A software programthat is particularly useful in homology modeling includes XALIGN(Wishart, D. et al., (1994) Cabios 10: 687-88). See also, U.S. Pat. No.5,884,230.

To model the rRNA of a new species, bases of the H. marismortui rRNA canbe replaced, using a computer graphics program such as “O” (Jones etal., (1991) Acta Cryst. Sect. A, 47: 110-119), by those of thehomologous rRNA, where they differ. In many if not most cases the sameorientation of the base will be appropriate. Insertions and deletionsmay be more difficult and speculative, but the rRNA forming the peptidyltransferase site and the portion of the tunnel closest to it is veryhighly conserved with essentially no insertions and deletions. Automatedweb-based homology modeling can be performed using, for example, thecomputer programs SWISS-MODEL available through Glaxo WelcomeExperimental Research in Geneva, Switzerland, and WHATIF available onEMBL servers.

For other descriptions of homology modeling, see, for example, Gutell R.R. (1996), supra; Gutell R. R., et al. (1993) Nucleic Acids Res. 21:3055-3074; Schnare et al. (1996) J. Mol. Biol., 256: 701-719; Blundellet al. (1987) Nature 326: 347-352; Fetrow and Bryant (1993)Bio/Technology 11:479-484; Greer (1991) Methods in Enzymology 202:239-252; and Johnson et al. (1994) Crit. Rev. Biochem. Mol. Biol.29:1-68. An example of homology modeling can be found, for example, inSzklarz G. D (1997) Life Sci. 61: 2507-2520.

As discussed earlier, the large ribosomal subunit from prokaryotes andeukaryotes and eukaryotic mitochondria are structurally conserved. Theamino acid sequences of the large ribosomal subunit from prokaryotes andeukaryotes can be aligned due to the evolutionary conservation of aminoacid residues that are important for three-dimensional structure, thenature and shape of the binding sites for substrates and the catalyticsite. This similarity in amino acid sequence of the homologous largeribosomal subunit allows the construction of models, via homologymodeling, for the molecules whose crystal structures have not beensolved.

The new ribosome or large ribosomal subunit structures determined usingthe H. marismortui crystals and/or atomic co-ordinates can then be usedfor structure-based drug design using one or more of the approachesdescribed hereinbelow. This information can then be used to designmolecules that selectively bind and disrupt protein synthesis in theribosomes of the pathogens while leaving the ribosomes of a hostrelatively unaffected.

G. Rational Drug Design

1. Introduction

It is contemplated that the atomic co-ordinates defining a largeribosomal subunit of interest, whether derived from one or more of X-raycrystallography, molecular modeling, homology modeling or molecularreplacement, may be used in rational drug design (RDD) to design a novelmolecule of interest, for example, novel modulators (for example,inducers, mimetics or inhibitors) of ribosome function. Furthermore, itis contemplated that, by using the principles disclosed herein, theskilled artisan can design, make, test, refine and use novel proteinsynthesis inhibitors specifically engineered to reduce, disrupt, orotherwise or inhibit ribosomal function in an organism or species ofinterest. For example, by using the principles discussed herein, theskilled artisan can engineer new molecules that specifically target andinhibit ribosomal function in a pathogen, for example, a particularprokaryotic, organism, while preserving ribosomal function in a host,for example, a eukaryotic organism, specifically a mammal, and morespecifically, a human. As a result, the atomic co-ordinates provided anddiscussed herein permit the skilled artisan to design new antibioticsthat can kill certain pathogenic organisms while having little or notoxicity in the intended recipient, for example, a human.

It is contemplated that RDD using atomic co-ordinates of the largeribosomal subunit can be facilitated most readily via computer-assisteddrug design (CADD) using conventional computer hardware and softwareknown and used in the art. The candidate molecules may be designed denovo or may be designed as a modified version of an already existingmolecule, for example, a pre-existing antibiotic, using conventionalmethodologies. Once designed, candidate molecules can be synthesizedusing standard methodologies known and used in the art. Followingsynthesis, the candidate molecules can be screened for bioactivity, forexample, by their ability to reduce or inhibit ribosome function, theirability to interact with or bind a ribosome or a ribosomal subunit.Based in part upon these results, the candidate molecules may be refinediteratively using one or more of the foregoing steps to produce a moredesirable molecule with a desired biological activity. The resultingmolecules can be useful in treating, inhibiting or preventing thebiological activities of target organisms, thereby killing the organismor impeding its growth. Alternatively, the resulting molecules can beuseful for treating, inhibiting or preventing microbial infections inany organism, particularly animals, more particularly humans.

In summary, the tools and methodologies provided by the presentinvention may be used to identify and/or design molecules which bindand/or interact in desirable ways with ribosomes and ribosomal subunits.Basically, the procedures utilize an iterative process whereby themolecules are synthesized, tested and characterized. New molecules canbe designed based on the information gained in the testing andcharacterization of the initial molecules and then such newly identifiedmolecules can themselves be tested and characterized. This series ofprocesses may be repeated as many times as necessary to obtain moleculeswith desirable binding properties and/or biological activities. Methodsfor identifying candidate molecules are discussed in more detail below.

2. Identification of Candidate Molecules

It is contemplated that the design of candidate molecules of interestcan be facilitated by conventional ball and stick-type modelingprocedures. However, in view of the size and complexity of the largeribosomal subunit, it is contemplated that the ability to designcandidate molecules may be enhanced significantly using computer-basedmodeling and design protocols.

a. Molecular Modeling.

It is contemplated that the design of candidate molecules, as discussedin detail hereinbelow, can be facilitated using conventional computersor workstations, available commercially from, for example, SiliconGraphics Inc. and Sun Microsystems, running, for example, UNIX based,Windows NT on IBM OS/2 operating systems, and capable of runningconventional computer programs for molecular modeling and rational drugdesign.

It is understood that any computer system having the overallcharacteristics set forth in FIG. 31 may be useful in the practice ofthe invention. More specifically, FIG. 31, is a schematic representationof a typical computer work station having in electrical communication(100) with one another via, for example, an internal bus or externalnetwork, a central processing unit (101), a random access memory (RAM)(102), a read only memory (ROM) (103), a monitor or terminal (104), andoptimally an external storage device, for example, a diskette, CD ROM,or magnetic tape (105).

The computer-based systems of the invention preferably comprise a datastorage means having stored therein a ribosome or ribosomal subunit orfragment sequence and/or atomic co-ordinate/X-ray diffraction data ofthe present invention and the necessary hardware means and softwaremeans for supporting and implementing an analysis means. As used herein,“a computer system” or “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thesequence, X-ray diffraction data, and/or atomic co-ordinates of theinvention. As used herein, the term “data storage means” is understoodto refer to any memory which can store sequence data, atomicco-ordinates, and/or X-ray diffraction data, or a memory access meanswhich can access manufactures having recorded thereon the atomicco-ordinates of the present invention.

In one embodiment, a ribosome or ribosomal subunit, or at least asubdomain thereof, amino acid and nucleic acid sequence, X-raydiffraction data and/or atomic co-ordinates of the present invention arerecorded on computer readable medium. As used herein, the term “computerreadable medium” is understood to mean any medium which can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedium, and magnetic tape; optical storage media such as optical discsor CD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media. A skilledartisan can readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising computerreadable medium having recorded thereon an amino acid and/or nucleotidesequence, X-ray diffraction data, and/or atomic co-ordinates of thepresent invention.

As used herein, the term “recorded” is understood to mean any processfor storing information on computer readable medium. A skilled artisancan readily adopt any of the presently known methods for recordinginformation on computer readable medium to generate manufacturescomprising an amino acid or nucleotide sequence, atomic co-ordinatesand/or X-ray diffraction data of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon aminoacid and/or nucleotide sequence, atomic co-ordinates and/or X-raydiffraction data of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the sequence information,X-ray data and/or atomic co-ordinates of the present invention oncomputer readable medium. The foregoing information, data andco-ordinates can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMICROSOFT Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of data processorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the information of thepresent invention.

By providing a computer readable medium having stored thereon a ribosomeor ribosomal subunit sequence, and/or atomic co-ordinates, a skilledartisan can routinely access the sequence, and/or atomic co-ordinates tomodel a ribosome or ribosomal subunit, a subdomain thereof, mimetic, ora ligand thereof Computer algorithms are publicly and commerciallyavailable which allow a skilled artisan to access this data provided ina computer readable medium and analyze it for molecular modeling and/orRDD. See, e.g., Biotechnology Software Directory, MaryAnn Liebert Publ.,New York, N.Y. (1995).

Although computers are not required, molecular modeling can be mostreadily facilitated by using computers to build realistic models of aribosome, ribosomal subunit, or a portion thereof. Molecular modelingalso permits the modeling of new smaller molecules, for example ligands,agents and other molecules, that can bind to a ribosome, ribosomalsubunit, or a portion therein. The methods utilized in molecularmodeling range from molecular graphics (i.e., three-dimensionalrepresentations) to computational chemistry (i.e., calculations of thephysical and chemical properties) to make predictions about the bindingof the smaller molecules or their activities; to design new molecules;and to predict novel molecules, including ligands such as drugs, forchemical synthesis.

For basic information on molecular modeling, see, for example, M.Schlecht, Molecular Modeling on the PC (1998) John Wiley & Sons; Gans etal., Fundamental Principals of Molecular Modeling (1996) Plenum Pub.Corp.; N.C. Cohen, ed., Guidebook on Molecular Modeling in Drug Design(1996) Academic Press; and W. B. Smith, Introduction to TheoreticalOrganic Chemistry and Molecular Modeling (1996). U.S. Patents whichprovide detailed information on molecular modeling include, for example:U.S. Pat. Nos. 6,093,573; 6,080,576; 6,075,014; 6,075,123; 6,071,700;5,994,503; 5,884,230; 5,612,894; 5,583,973; 5,030,103; 4,906,122; and4,812,12.

Three-dimensional modeling can include, but is not limited to, makingthree-dimensional representations of structures, drawing pictures ofstructures, building physical models of structures, and determining thestructures of related ribosomes, ribosomal subunits and ribosome/ligandand ribosomal subunit/ligand complexes using the known co-ordinates. Theappropriate co-ordinates are entered into one or more computer programsfor molecular modeling, as known in the art. By way of illustration, alist of computer programs useful for viewing or manipulatingthree-dimensional structures include: Midas (University of California,San Francisco); MidasPlus (University of California, San Francisco);MOIL (University of Illinois); Yummie (Yale University); Sybyl (Tripos,Inc.); Insight/Discover (Biosym Technologies); MacroModel (ColumbiaUniversity); Quanta (Molecular Simulations, Inc.); Cerius (MolecularSimulations, Inc.); Alchemy (Tripos, Inc.); LabVision (Tripos, Inc.);Rasmol (Glaxo Research and Development); Ribbon (University of Alabama);NAOMI (Oxford University); Explorer Eyechem (Silicon Graphics, Inc.);Univision (Cray Research); Molscript (Uppsala University); Chem-3D(Cambridge Scientific); Chain (Baylor College of Medicine); O (UppsalaUniversity); GRASP (Columbia University); X-Plor (Molecular Simulations,Inc.; Yale University); Spartan (Wavefunction, Inc.); Catalyst(Molecular Simulations, Inc.); Molcadd (Tripos, Inc.); VMD (Universityof Illinois/Beckman Institute); Sculpt (Interactive Simulations, Inc.);Procheck (Brookhaven National Library); DGEOM (QCPE); RE_VIEW (BrunellUniversity); Modeller (Birbeck College, University of London); Xmol(Minnesota Supercomputing Center); Protein Expert (CambridgeScientific); HyperChem (Hypercube); MD Display (University ofWashington); PKB (National Center for Biotechnology Information, NIH);ChemX (Chemical Design, Ltd.); Cameleon (Oxford Molecular, Inc.); andIditis (Oxford Molecular, Inc.).

One approach to RDD is to search for known molecular structures thatmight bind to a site of interest. Using molecular modeling, RDD programscan look at a range of different molecular structures of molecules thatmay fit into a site of interest, and by moving them on the computerscreen or via computation it can be decided which structures actuallyfit the site well (William Bains (1998) Biotechnology from A to Z,second edition, Oxford University Press, p. 259).

An alternative but related approach starts with the known structure of acomplex with a small molecule ligand and models modifications of thatsmall molecule in an effort to make additional favorable interactionswith a ribosome or ribosomal subunit.

The present invention permits the use of molecular and computer modelingtechniques to design and select novel molecules, such as antibiotics orother therapeutic agents, that interact with ribosomes and ribosomalsubunits. Such antibiotics and other types of therapeutic agentsinclude, but are not limited to, antifungals, antivirals,antibacterials, insecticides, herbicides, miticides, rodentcides, etc.

In order to facilitate molecular modeling and/or RDD the skilled artisanmay use some or all of the atomic co-ordinates deposited at the RCSBProtein Data Bank with the accession number PDB ID: 1FFK, 1JJ2, 1FFZ,1FG0, 1K73, 1KC8, 1K8A, 1KD1, or 1K9M, atomic co-ordinates contained onDisk No. 1. Furthermore, the skilled artisan, using the foregoing atomicco-ordinates, the skilled artisan can generate additional atomicco-ordinates via, for example, molecular modeling using, for example,homology modeling and/or molecular replacement techniques, that togetherdefine at least a portion of a model of a ribosome from another speciesof interest. By using the foregoing atomic co-ordinates, the skilledartisan can design inhibitors of protein synthesis that may be tailoredto be effective against ribosomes from one or more species but whichhave little or no effect on ribosomes of other species. Such inhibitorsmay be competitive inhibitors. As used herein, the term “competitiveinhibitor” refers to an inhibitor that binds to the active form of aribosome or ribosomal subunit at the same sites as its substrate(s) ortRNA(s), thus directly competing with them. The term “active form” of aribosome or ribosomal subunit refers to a ribosome or ribosomal subunitin a state that renders it capable of protein synthesis. Competitiveinhibition can be reversed completely by increasing the substrate ortRNA concentration.

This invention also permits the design of molecules that act asuncompetitive inhibitors of protein synthesis. As used herein, the term“uncompetitive inhibitor” refers to a molecule that inhibits thefunctional activity of a ribosome or ribosomal subunit by binding to adifferent site on the ribosome or ribosomal subunit than does itssubstrates, or tRNA. Such inhibitors can often bind to the ribosome orribosomal subunit with the substrate or tRNA and not to the ribosome orribosomal subunit by itself. Uncompetitive inhibition cannot be reversedcompletely by increasing the substrate concentration. These inhibitorsmay bind to, all or a portion of, the active sites or other regions ofthe large ribosomal subunit already bound to its substrate and may bemore potent and less non-specific than known competitive inhibitors thatcompete for large ribosomal subunit active sites or for binding to largeribosomal subunit.

Similarly, non-competitive inhibitors that bind to and inhibit proteinsynthesis whether or not it is bound to another chemical entity may bedesigned using the atomic co-ordinates of the large ribosomal subunitsor complexes comprising large ribosomal subunit of this invention. Asused herein, the term “non-competitive inhibitor” refers to an inhibitorthat can bind to either the free or substrate or tRNA bound form of theribosome or ribosomal subunit.

Those of skill in the art may identify inhibitors as competitive,uncompetitive, or non-competitive by computer fitting enzyme kineticdata using standard equation according to Segel, I. H., (1975) EnzymeKinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-StateEnzyme Systems, (Wiley Classics Library). It should also be understoodthat uncompetitive or non-competitive inhibitors according to thepresent invention may bind the same or different binding sites.

Alternatively, the atomic co-ordinates provided by the present inventionare useful in designing improved analogues of known protein synthesisinhibitors or to design novel classes of inhibitors based on the atomicstructures and co-ordinates of the crystals of the 50S ribosomalsubunit/CCdA-p-Puro complex and the 50S ribosomal subunit/aa-tRNAanalogue complex. This provides a novel route for designing inhibitorsof protein synthesis with both high specificity, stability and otherdrug-like qualities (Lipinski et al. (1997) Adv. Drug Deliv. Rev. 23:3).

The atomic co-ordinates of the present invention also permit probing thethree-dimensional structure of a ribosome or ribosome subunit or aportion thereof with molecules composed of a variety of differentchemical features to determine optimal sites for interaction betweencandidate inhibitors and/or activators and the ribosome or ribosomalsubunit. For example, high resolution atomic co-ordinates based on X-raydiffraction data collected from crystals saturated with solvent allowsthe determination of where each type of solvent molecule sticks. Smallmolecules that bind to those sites can then be designed and synthesizedand tested for their inhibitory activity (Travis, J. (1993) Science 262:1374). Further, any known antibiotic, inhibitor or other small moleculethat binds to the H. marismortui large subunit can be soaked into H.marismortui large subunit crystals and their exact mode of bindingdetermined from difference electron density maps. These molecules mayrepresent lead compounds from which better drug-like compounds can besynthesized.

b. Identification of Target Sites.

The atomic co-ordinates of the invention permit the skilled artisan toidentify target locations in a ribosome or large ribosomal subunit thatcan serve as a starting point in rational drug design. As a thresholdmatter, the atomic co-ordinates of the invention permit the skilledartisan to identify specific regions within a ribosome or ribosomalsubunit that are involved with protein synthesis and/or proteinsecretion out of the ribosome. Furthermore, the atomic co-ordinates ofthe invention permit a skilled artisan to further identify portions ofthese regions that are conserved or are not conserved between differentorganisms. For example, by identifying portions of these regions thatare conserved among certain pathogens, for example, certain prokaryotes,but are not conserved in a host organism, for example, a eukaryote, morepreferably a mammal, the skilled artisan can design molecules thatselectively inhibit or disrupt protein synthesis activity of thepathogen's but not the host's ribosomes. Furthermore, by analyzingregions that are either conserved or non-conserved between certainpathogens, it may be possible to design broad or narrow spectrum proteinsynthesis inhibitors, e.g., antibiotics, as a particular necessityarises.

FIG. 32, is a schematic representation of a large ribosomal subunit thatidentifies a variety of exemplary target sites that appear toparticipate in protein synthesis within the ribosome and/or the exportor translocation of the newly synthesized protein out of the ribosome.The target sites include, for example, the P-site (200), the A-site(201), the peptidyl transferase center (202), the peptidyl transferasesite (203) which includes at least a portion of the P-site and theA-site, a factor binding domain (204) including, for example, the EF-Tubinding domain and the EF-G binding domain, the polypeptide exit tunnel(205) including cavities defined by the wall of the exit tunnel, and thesignal recognition particle binding domain (206).

By way of example, inspection of the atomic co-ordinates of the Hmarismortui 50S ribosomal subunit has identified a variety of targetregions that may serve as a basis for the rational drug design of new ormodified protein synthesis inhibitors. The target regions include thepeptidyl transferase site, A-site, the P-site, the polypeptide exittunnel, certain cavities disposed in the wall of the polypeptide exittunnel (for example, cavity 1 and cavity 2), and certain antibioticbinding pockets. The residues that together define at least a portion ofeach of the foregoing regions are identified in the following tables.However, it is contemplated that the same or similar target sites can beidentified in a ribosome or a ribosomal unit of interest using theprinciples described herein. Furthermore, these principles can beemployed using any of the primary sets of atomic co-ordinates providedherein or any additional atomic co-ordinate sets, for example, secondaryatomic co-ordinate sets that may be generated by molecular modeling ofany ribosome or ribosomal subunit of interest.

Table 5A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of the ribosomalpeptidyl transferase site (5.8 Å shell). In addition, Table 5Aidentifies the corresponding residues that define at least a portion ofthe ribosomal peptidyl transferase site in E coli, Rattus, human, andhuman mitochondria large subunit. Table 5B identifies the residues inthe H. marismortui 50S ribosomal subunit that together define a broaderportion of the ribosomal peptidyl transferase site (5.8 Å-12.6 Å shell).The non-conserved residues were identified by comparison of sequencesfrom the structure of H. marismortui 23S rRNA or ribosomal protein thatform the above-mentioned sites with the corresponding sequences ofaligned genomic DNA encoding either the homologous 23S rRNA or ribosomalprotein from the other organisms.

TABLE 5A Residues that Define the Ribosomal Peptidyl-Transferase Site(5.8 Å shell) Corre- Corre- Corresponding sponding spondingCorresponding Residue in H. marismortui Residue in Residue Residue inHuman Residue E. coli in Rattus Human Mitochondria 23S rRNA G2102 G2061G3632 G3876 G1054 A2103 A2062 A3633 A3877 A1055 C2104 C2063 C3634 C3878C1056 C2105 C2064 C3635 C3879 C1057 C2106 C2065 C3636 C3880 C1058 G2284G2251 G3917 G4156 G1145 G2285 G2252 G3918 G4157 G1146 G2286 G2253 G3919G4158 G1147 A2474 A2439 A4106 A4345 A1256 G2482 G2447 G4114 G4353 G1264A2485 A2450 A4117 A4356 A1267 A2486 A2451 A4118 A4357 A1268 C2487 C2452C4119 C4358 C1269 A2488 A2453 U4120 U4359 A1270 U2528 U2493 U4160 U4399U1310 G2529 G2494 G4161 G4400 A1311 C2536 C2501 C4168 C4407 C1318 A2538A2503 A4170 A4409 A1320 G2540 G2505 G4172 G4411 G1322 U2541 U2506 U4173U4412 U1323 C2542 C2507 C4174 C4413 U1324 G2543 G2508 G4175 G4414 G1325G2588 G2553 G4220 G4459 G1370 U2589 U2554 U4221 U4460 U1371 U2590 U2555U4222 U4461 U1372 C2608 C2573 C4240 C4479 C1390 G2617 G2582 G4249 G4488A1399 G2618 G2583 G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401 U2620U2585 U4252 U4491 U1402 C2636 C2601 C4268 C4507 C1418 A2637 A2602 A4269A4508 A1419 G2638 G2603 G4270 G4509 G1420 Residues that together definethe peptidyl transferase site (5.8 Å shell) were determined to be thoseresidues in the 50S ribosomal subunit that are within 5.8 angstroms ofthe atoms of the # CC-Puromycin A-site ligand and the atoms of theCCdA-PO₂ moiety of the CCdA-p-Puromycin transition state inhibitor (PDBaccession codes: 1 fg0 and 1 ffz, respectively) using the programMIDAS^(a). Conserved # residues in 23S rRNA were determined bycomparison of sequences from the H. marismortui 23S rRNA^(b) withgenomic DNA sequences encoding the homologous 23S rRNA (E. coli ^(c),Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)). Sequence #alignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683

TABLE 5B Residues that Define the Ribosomal Peptidyl-Transferase Site(5.8 Å-12.6 Å shell) Corre- Corre- Corresponding sponding spondingCorresponding Residue in H. marismortui Residue in Residue Residue inHuman Residue E. coli in Rattus Human Mitochondria 23S rRNA C1750 A1672C2692 C2830 A732 C1982 C1941 C3513 C3757 A934 C1983 C1942 C3514 C3758C935 U1984 U1943 U3515 U3759 U936 U1985 U1944 C3516 C3760 U937 U1996U1955 U3527 U3771 U948 C2006 C1965 C3537 C3781 U958 A2007 A1966 A3538A3782 A959 G2073 G2032 C3603 C3847 G1025 A2100 A2059 A3630 A3874 A1052A2103 A2062 A3633 A3877 A1055 U2107 C2066 U3637 U3881 U1059 U2282 U2249U3915 U4154 U1143 G2283 G2250 G3916 G4155 G1144 C2287 C2254 C3920 C4159C1148 G2288 G2255 G3921 G4160 G1149 C2309 C2275 C3942 C4181 C1154 C2472G2437 U4104 U4343 U1254 U2473 U2438 U4105 U4344 U1255 C2476 U2441 C4108C4347 C1258 A2479 G2444 A4111 A4350 A1261 G2480 G2445 G4112 G4351 G1262G2481 G2446 G4113 G4352 G1263 G2482 G2447 G4114 G4353 G1264 U2484 U2449U4116 U4355 U1266 G2489 G2454 G4121 G4360 G1271 A2490 G2455 G4122 G4361C1272 C2526 U2491 U4158 U4397 U1308 U2527 U2492 U4159 U4398 U1309 G2529G2494 G4161 G4400 A1311 C2530 G2495 A4162 A4401 C1312 U2531 C2496 U4163U4402 G1313 A2532 A2497 C4164 C4403 A1314 C2534 C2499 U4166 U4405 C1316U2535 U2500 U4167 U4406 U1317 G2537 G2502 G4169 G4408 G1319 A2538 A2503A4170 A4409 A1320 U2539 U2504 U4171 U4410 U1321 G2540 G2505 G4172 G4411G1322 G2544 G2509 G4176 G4415 G1326 U2587 U2552 U4219 U4458 U1369 C2591C2556 C4223 C4462 C1373 G2592 G2557 A4224 A4463 A1374 A2604 G2569 G4236G4475 C1386 G2605 G2570 G4237 G4476 C1387 U2607 A2572 A4239 A4478 A1389G2609 G2574 G4241 G4480 G1391 U2610 C2575 U4242 U4481 U1392 G2611 G2576G4243 G4482 G1393 G2616 G2581 G4248 G4487 G1398 G2617 G2582 G4249 G4488A1399 U2621 U2586 U4253 U4492 C1403 A2622 A2587 A4254 A4493 A1404 G2627G2592 G4259 G4498 G1409 U2628 U2593 U4260 U4499 G1410 G2634 G2599 G4266G4505 U1416 A2635 A2600 A4267 A4506 C1417 G2638 G2603 G4270 G4509 G1420G2639 U2604 G4271 G4510 G1421 U2640 U2605 U4272 U4511 U1422 G2643 G2608G4275 G4514 G1425 U2645 C2610 U4277 U4516 U1427 Protein L3 P1 NP* S2 S2P2 Protein L10E D109 XX** T113 T113 XX G110 XX G114 G114 XX R112 XX R116R116 XX Residues that together define the peptidyl transferase site (5.8Å-12.6 Å shell) were determined to be those residues in the 50Sribosomal subunit that are within 5.8-12.6 angstroms of the atoms of theCC-Puromycin A-site ligand and the atoms of the CCdA-PO₂ moiety # of theCCdA-p-Puromycin transition state inhibitor (PDB accession codes: 1 fg0and 1 ffz, respectively), using the program MIDAS^(a). Conservedresidues in 23S rRNA were determined by comparison of sequences from thestructure of H. marismortui 23S rRNA^(b) with the # correspondingsequences of aligned genomic DNA encoding the homologous 23S rRNA (E.coli ^(c), Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)).In cases of ribosomal proteins, comparisons were also made between theH. marismortui structural # protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were determined with the program #MegAlign (DNASTAR, Madison, Wisconsin, USA) using defaul parameters. *NPmeans that no homologous residue has been identified. **XX means that nohomologous protein has been identified in that species. ^(a)T. E.Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDASDisplay System” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBankaccession AF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession 13683^(g)GenBank accession AAA86859 for protein L3; GenBank accession15825950 for protein L10E. ^(i)GenBank accession CAA26460 for proteinL3. ^(j)GenBank accession P21531 for protein L3; GenBank accessionNP_112362 for protein L10E. ^(k)GenBank accession NP_000958 for proteinL3; GenBank accession NP_006004 for protein L10E. ^(l)GenBank accessionP09001 for protein L3.

Table 6A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of the ribosomal A-site(5.8 Å shell). In addition, Table 6A identifies the correspondingresidues that define at least a portion of the ribosomal A-site in E.coli, Rattus, human, and human mitochondria large subunit. Table 6Bidentifies the residues in the H. marismortui 50S ribosomal subunit thattogether define a broader portion of the ribosomal A-site (5.8 Å-12.6 Åshell). The non conserved residues were identified as describedpreviously with respect to Tables 5A and 5B.

TABLE 6A Residues that Define the Ribosomal A-site (5.8 Å shell) Corre-Corre- Corresponding sponding sponding Corresponding Residue in H.marismortui Residue in Residue Residue in Human Residue E. coli inRattus Human Mitochondria 23S rRNA G2102 G2061 G3632 G3876 G1054 C2104C2063 C3634 C3878 C1056 A2485 A2450 A4117 A4356 A1267 A2486 A2451 A4118A4357 A1268 C2487 C2452 C4119 C4358 C1269 A2488 A2453 U4120 U4359 A1270U2528 U2493 U4160 U4399 U1310 G2529 G2494 G4161 G4400 A1311 C2536 C2501C4168 C4407 C1318 U2541 U2506 U4173 U4412 U1323 C2542 C2507 C4174 C4413U1324 G2543 G2508 G4175 G4414 G1325 G2588 G2553 G4220 G4459 G1370 U2589U2554 U4221 U4460 U1371 U2590 U2555 U4222 U4461 U1372 C2608 C2573 C4240C4479 C1390 G2618 G2583 G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401U2620 U2585 U4252 U4491 U1402 Residues that together define the A-site(5.8 Å shell) were determined to be those residues in the 50S ribosomalsubunit that are within 5.8 angstroms of the atoms of the CC-PuromycinA-site ligand (PDB accession code 1 fg0) using the program MIDAS^(a).Conserved # residues in 23S rRNA were determined by comparison ofsequences from the structure of H. marismortui 23S rRNA^(b) with thecorresponding sequences of aligned genomic DNA encoding the homologous23S rRNA (E. coli ^(c), Rattus norvegicus ^(d), Human^(e) or Human #mitochondria^(f)). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988)“The MIDAS Display System” J. Mol. Graphics, 6(1):13-27, 36-37.^(b)GenBank accession AF034619 ^(c)GenBank accession J01695 ^(d)GenBankaccession 2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession13683

TABLE 6B Residues that Define the Ribosomal A-site (5.8 Å-12.6 Å shell)Corre- Corre- Corresponding sponding sponding Corresponding Residue inH. marismortui Residue in Residue Residue in Human Residue E. coli inRattus Human Mitochondria 23S rRNA C1750 A1672 C2692 C2830 A732 C1982C1941 C3513 C3757 A934 C1983 C1942 C3514 C3758 C935 U1984 U1943 U3515U3759 U936 U1985 U1944 C3516 C3760 U937 U1996 U1955 U3527 U3771 U948G2073 G2032 C3603 C3847 G1025 A2100 A2059 A3630 A3874 A1052 A2103 A2062A3633 A3877 A1055 C2105 C2064 C3635 C3879 C1057 C2106 C2065 C3636 C3880C1058 U2107 C2066 U3637 U3881 U1059 G2284 G2251 G3917 G4156 G1145 G2285G2252 G3918 G4157 G1146 A2474 A2439 A4106 A4345 A1256 A2479 G2444 A4111A4350 A1261 G2480 G2445 G4112 G4351 G1262 G2481 G2446 G4113 G4352 G1263G2482 G2447 G4114 G4353 G1264 U2484 U2449 U4116 U4355 U1266 G2489 G2454G4121 G4360 G1271 A2490 G2455 G4122 G4361 C1272 C2526 U2491 U4158 U4397U1308 U2527 U2492 U4159 U4398 U1309 C2530 G2495 A4162 A4401 C1312 U2531C2496 U4163 U4402 G1313 A2532 A2497 C4164 C4403 A1314 C2534 C2499 U4166U4405 C1316 U2535 U2500 U4167 U4406 U1317 G2537 G2502 G4169 G4408 G1319A2538 A2503 A4170 A4409 A1320 U2539 U2504 U4171 U4410 U1321 G2540 G2505G4172 G4411 G1322 G2544 G2509 G4176 G4415 G1326 U2587 U2552 U4219 U4458U1369 C2591 C2556 C4223 C4462 C1373 G2592 G2557 A4224 A4463 A1374 A2604G2569 G4236 G4475 C1386 G2605 G2570 G4237 G4476 C1387 U2607 A2572 A4239A4478 A1389 G2609 G2574 G4241 G4480 G1391 U2610 C2575 U4242 U4481 U1392G2611 G2576 G4243 G4482 G1393 G2616 G2581 G4248 G4487 G1398 G2617 G2582G4249 G4488 A1399 U2621 U2586 U4253 U4492 C1403 C2636 C2601 C4268 C4507C1418 A2637 A2602 A4269 A4508 A1419 G2638 G2603 G4270 G4509 G1420 G2639U2604 G4271 G4510 G1421 U2640 U2605 U4272 U4511 U1422 G2643 G2608 G4275G4514 G1425 U2645 C2610 U4277 U4516 U1427 Protein L3 P1 NP* S2 S2 P2Residues that define the A-site (5.8 Å-12.6 Å shell) were determined tobe those residues in the 50S ribosomal subunit that are within 5.8-12.6angstroms of the atoms of the CC-Puromycin A-site ligand (PDB accessioncode 1 fg0) using the # program MIDAS^(a). Conserved residues in 23SrRNA were determined by comparison of sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). In cases ofribosomal # proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) with default parameters. *NPmeans that no homologous residue has been identified. ^(a)T. E. Ferrin,C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDAS DisplaySystem” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBank accessionAF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession 2624399^(e)GenBank accession M11167 ^(f)GenBank accession 13683 ^(g)GenBankaccession AAA86859 for protein L3. ^(i)GenBank accession CAA26460 forprotein L3. ^(j)GenBank accession P21531 for protein L3. ^(k)GenBankaccession NP_000958 for protein L3. ^(l)GenBank accession P09001 forprotein L3.

Table 7A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of the ribosomal P-site(5.8 Å shell). In addition, Table 7A identifies the correspondingresidues that define at least a portion of the ribosomal P-site in E.coli, Rattus, human, and human mitochondria large subunit. Table 7Bidentifies the residues in the H. marismortui 50S ribosomal subunit thattogether define a broader portion of the ribosomal P-site (5.8 Å-12.6 Åshell). The non conserved residues were identified as describedpreviously with respect to Tables 5A and 5B.

TABLE 7A Residues that Define the Ribosomal P-site (5.8 Å shell) Corre-Corre- Corresponding sponding sponding Corresponding Residue in H.marismortui Residue in Residue Residue in Human Residue E. coli inRattus Human Mitochondria 23S rRNA C2104 C2063 C3634 C3878 C1056 C2105C2064 C3635 C3879 C1057 C2106 C2065 C3636 C3880 C1058 G2284 G2251 G3917G4156 G1145 G2285 G2252 G3918 G4157 G1146 G2286 G2253 G3919 G4158 G1147A2474 A2439 A4106 A4345 A1256 A2485 A2450 A4117 A4356 A1267 A2486 A2451A4118 A4357 A1268 U2619 U2584 U4251 U4490 U1401 U2620 U2585 U4252 U4491U1402 C2636 C2601 G4268 C4507 C1418 A2637 A2602 A4269 A4508 A1419 G2638G2603 G4270 G4509 G1420 Residues that together define the P-site (5.8 Åshell) were determined to be those residues in the 50S ribosomal subunitthat are within 5.8 angstroms of the atoms of the CCdA-PO₂ moiety of theCCdA-p-puromycin transition state inhibitor (FIG. 10a) (PDB accessioncode 1 ffz) using the program MIDAS^(a). Conserved residues in 23S rRNAwere determined by comparison of # sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). Sequencealignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683

TABLE 7B Residues that Define the Ribosomal P-site (5.8 Å-12.6 Å shell)Corre- Corre- Corresponding sponding sponding Corresponding Residue inH. marismortui Residue in Residue Residue in Human Residue E. coli inRattus Human Mitochondria 23S rRNA C1982 C1941 C3513 C3757 A934 C2006C1965 C3537 C3781 U958 A2007 A1966 A3538 A3782 A959 G2102 G2061 G3632G3876 G1054 A2103 A2062 A3633 A3877 A1055 U2107 C2066 U3637 U3881 U1059U2282 U2249 U3915 U4154 U1143 G2283 G2250 G3916 G4155 G1144 C2287 C2254C3920 C4159 C1148 G2288 G2255 G3921 G4160 G1149 C2309 C2275 C3942 C4181C1154 C2472 G2437 U4104 U4343 U1254 U2473 U2438 U4105 U4344 U1255 C2476U2441 C4108 C4347 C1258 G2481 G2446 G4113 G4352 G1263 G2482 G2447 G4114G4353 G1264 U2484 U2449 U4116 U4355 U1266 C2487 C2452 C4119 C4358 C1269A2488 A2453 U4120 U4359 A1270 U2528 U2493 U4160 U4399 U1310 G2529 G2494G4161 G4400 A1311 C2530 G2495 A4162 A4401 C1312 U2531 C2496 U4163 U4402G1313 A2532 A2497 C4164 C4403 A1314 C2534 C2499 U4166 U4405 C1316 U2535U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407 C1318 A2538 A2503 A4170A4409 A1320 G2540 G2505 G4172 G4411 G1322 U2541 U2506 U4173 U4412 U1323C2542 C2507 C4174 C4413 U1324 C2608 C2573 C4240 C4479 C1390 G2611 G2576G4243 G4482 G1393 G2618 G2583 G4250 G4489 G1400 U2621 U2586 U4253 U4492C1403 A2622 A2587 A4254 A4493 A1404 G2627 G2592 G4259 G4498 G1409 U2628U2593 U4260 U4499 G1410 G2634 G2599 G4266 G4505 U1416 A2635 A2600 A4267A4506 C1417 G2639 U2604 G4271 G4510 G1421 U2640 U2605 U4272 U4511 U1422G2643 G2608 G4275 G4514 G1425 Protein L10E D109 XX** T113 T113 XX G110XX G114 G114 XX R112 XX R116 R116 XX Residues that together define theP-site (5.8 Å-12.6 Å shell) were determined to be those residues in the50S ribosomal subunit that are within the atoms of the CCdA-PO₂ moietyof the CCdA-p-puromycin transition state inhibitor (FIG. 10a) (PDBaccession code 1 ffz) using the program MIDAS^(a). Conserved residues in23S rRNA were determined by comparison of # sequences from the structureof H. marismortui 23S rRNA^(b) with the corresponding sequences ofaligned genomic DNA encoding the homologous 23S rRNA (E. coli ^(c),Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)). In casesof ribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and # the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.**XX means that no homologous protein has been identified in thatspecies. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge(1988) “The MIDAS Display System” J. Mol. Graphics, 6(1):13-27, 36-37.^(b)GenBank accession AF034619 ^(c)GenBank accession J01695 ^(d)GenBankaccession 2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession13683 ^(g)GenBank accession 15825950 for protein L10E. ^(i)No GenBankaccession number for protein L10E. ^(j)GenBank accession NP_112362 forprotein L10E. ^(k)GenBank accession NP_006004 for protein L10E. ^(l)NoGenBank accession number that compares to protein L10E.

Table 8A identifies the residues in the H. marismortui 50S ribosomalsubunit that are within 10 Å of a hypothetical, nascent polypeptidewithin the ribosomal exit tunnel (10 Å shell). In addition, Table 8Aidentifies the corresponding residues that define at least a portion ofthe ribosomal polypeptide exit tunnel in E. coli, Rattus, human, andhuman mitochondria large subunit. Further, Table 8B identifies theresidues in the H. marismortui 50S ribosomal subunit that that arebetween 10-15 Å of a hypothetical, nascent polypeptide within theribosomal exit tunnel (10 Å-15 Å shell). The non conserved residues wereidentified as described previously with respect to Tables 5A and 5B.

TABLE 8A Residues that Define the Ribosomal Peptide Exit Tunnel (10 Åshell) Corre- Corre- Corresponding sponding sponding CorrespondingResidue in H. marismortui Residue in Residue Residue in Human Residue E.coli in Rattus Human Mitochondria 23S rRNA A60 A64 C729 C682 NP* G88 G93G764 G713 NP G89 A94 G765 G714 NP A90 A95 C766 C715 NP A462 C456 C1112C1195 NP U465 U459 G1116 G1199 NP A466 A460 G1117 G1200 NP G467 C461G1118 G1201 NP U468 C462 G1119 A1202 NP G475 G469 U1127 U1208 NP A476A470 C1128 C1209 NP A477 A471 A1129 U1210 NP C478 A472 C1130 C1211 NPU488 A482 C1140 C1221 NP A497 G491 G1151 G1233 NP A498 A492 G1152 A1234NP A513 A507 A1167 G1249 NP G514 A508 A1168 A1250 NP A767 A676 A1425A1503 NP U835 U744 U1491 U1570 NP C839 U746 G1495 G1574 NP U840 U747U1496 U1575 NP A841 G748 G1497 G1576 NP A844 A751 A1500 A1579 NP U845A752 A1501 A1580 NP U1359 U1255 U2191 U2327 U351 C1360 G1256 C2192 C2328G352 C1361 C1257 U2193 U2329 U353 U1362 U1258 U2194 U2330 C354 G1363G1259 G2195 G2331 C355 G1364 A1260 G2196 G2332 A356 A1424 U1318 G2255G2391 G412 G1425 C1319 U2256 U2392 U413 C1426 C1320 G2257 G2393 A414A1427 A1321 A2258 A2394 A415 C1428 A1322 A2259 A2395 A416 U1429 C1323C2260 C2396 U417 G1430 G1324 A2261 A2397 NP C1439 G1333 A2270 A2406 U426U1440 G1334 C2271 C2407 A427 G1441 C1335 A2272 A2408 G428 A1442 A1336U2273 U2409 U429 A1689 A1614 C2635 C2771 A680 C1690 C1615 A2636 A2772U681 G1837 U1781 A3368 A3612 C831 U1838 U1782 U3369 U3613 C832 A2054A2013 A3585 A3829 A1006 A2055 A2014 A3586 A3830 A1007 C2056 A2015 A3587A3831 A1008 U2057 U2016 C3588 C3832 U1009 C2098 G2057 A3628 A3872 A1050G2099 A2058 G3629 G3873 G1051 A2100 A2059 A3630 A3874 A1052 A2101 A2060A3631 A3875 A1053 G2102 G2061 G3632 G3876 G1054 A2103 A2062 A3633 A3877A1055 C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879 C1057 A2474A2439 A4106 A4345 A1256 C2477 C2442 A4109 A4348 C1259 U2478 C2443 C4110C4349 U1260 G2481 G2446 G4113 G4352 G1263 G2482 G2447 G4114 G4353 G1264A2485 A2450 A4117 A4356 A1267 A2486 A2451 A4118 A4357 A1268 C2487 C2452C4119 C4358 C1269 A2488 A2453 U4120 U4359 A1270 C2536 C2501 C4168 C4407C1318 G2537 G2502 G4169 G4408 G1319 A2538 A2503 A4170 A4409 A1320 U2539U2504 U4171 U4410 U1321 G2540 G2505 G4172 G4411 G1322 U2541 U2506 U4173U4412 U1323 C2542 C2507 C4174 C4413 U1324 C2608 C2573 C4240 C4479 C1390G2611 G2576 G4243 G4482 G1393 G2616 G2581 G4248 G4487 G1398 G2618 G2583G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401 U2620 U2585 U4252 U4491U1402 U2621 U2586 U4253 U4492 C1403 A2637 A2602 A4269 A4508 A1419 C2644U2609 U4276 U4515 U1426 U2645 C2610 U4277 U4516 U1427 G2646 C2611 U4278U4517 U1428 C2647 C2612 U4279 U4518 C1429 Protein L4 E59 E51 E65 E65 R70S60 V52 S66 S66 G71 F61 T53 W67 W67 F72 G62 G54 G68 G68 E73 S63 S55 T69T69 Q74 G64 G56 G70 G70 E75 R65 K57 R71 R71 R76 G66 NP* A72 A72 G78 Q67NP V73 V73 L79 A68 NP A74 A74 A80 H69 K58 R75 R75 D81 V70 P59 I76 I76L82 P71 W60 P77 P77 H83 K72 R61 R78 R78 P84 L73 Q62 V79 V79 D85 D74 G64G81 G81 F87 G75 T65 G82 G82 A88 R76 G66 G83 G83 T89 A77 R67 T84 T84 A90Protein L22 E20 H9 N21 N21 Q73 E121 S81 K124 K124 P177 Q122 M82 M125M125 P178 Q123 K83 R126 R126 P179 G124 R84 R127 R127 P180 R125 I85 R128R128 E181 K126 M86 T129 T129 P182 P127 P87 Y130 Y130 P183 R128 R88 R131R131 K184 A129 A89 A132 A132 A186 M130 K90 H133 H133 V187 G131 G91 G134G134 A188 R132 R92 R135 R135 H189 A133 A93 I136 I136 A190 S134 D94 N137N137 K191 A135 R95 P138 P138 E192 W136 I96 Y139 Y139 Y193 N137 L97 M140M140 I194 Q140 T100 P143 P143 F197 Protein L39E N18 XX** N20 N20 N98 S19XX R21 R21 H106 R20 XX P22 P22 R107 V21 XX I23 I23 I108 P22 XX P24 P24G109 A23 XX Q25 Q25 D110 Y24 XX W26 W26 F111 V25 XX I27 I27 I112 M26 XXR28 R28 D113 L27 XX M29 M29 V114 K28 XX K30 K30 S115 T29 XX T31 T31 E116D30 XX G32 G32 G117 E31 XX N33 N33 P118 R35 XX Y37 Y37 H135 N36 XX N38N38 N136 H37 XX S39 S39 L137 K38 XX K40 K40 Q138 R39 XX R41 R41 R146 R40XX R42 R42 R147 H41 XX H43 H43 H156 R44 XX R46 R46 R170 N45 XX T47 T47S171 The residues in the 10 Å shell were determined to be those residuesin the 50S ribosomal subunit that are within 10 angstroms of the atomsof a model of a newly synthesized peptide positioned in the center ofthe exit tunnel using the program MIDAS^(a). Conserved residues in 23SrRNA were determined by comparison of sequences from the structure # ofH. marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). In cases ofribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and # the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.*NP means that no homologous residue has been identified. **XX meansthat no homologous protein has been identified in that species. ^(a)T.E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDASDisplay System” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBankaccession AF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession 13683^(g)GenBank accession P12735 for protein L4; GenBank accession R5HS22for protein L22; GenBank accession P22452 for protein L39E. ^(i)GenBankaccession CAA26461 for protein L4; GenBank accession CAA26465 forprotein L22; no GenBank accession for protein L39E. ^(j)GenBankaccession JC4277 for protein L4; GenBank accession P24049 for proteinL22; GenBank accession CAA57900 for protein L39E. ^(k)GenBank accessionsP36578, NP_00959, S39803, and T09551 for protein L4; GenBank accessionXP_057521 for protein L22; GenBank accession NP_000991 for protein L39E.^(l)GenBank accession XP_049502 for protein L4; GenBank accessionXP_051279 for protein L22; GenBank accession NP_059142 and NP_542984 forprotein L39E.

TABLE 8B Residues that Define the Ribosomal Peptide Exit Tunnel (10 Å-15Å shell) Corre- Corre- Corresponding sponding sponding CorrespondingResidue in H. marismortui Residue in Residue Residue in Human Residue E.coli in Rattus Human Mitochondria 23S rRNA U22 U25 G686 G637 NP* G23 G26G687 G638 NP G24 G27 G688 G639 NP C57 C61 C726 U679 NP C58 U62 U727 U680NP A59 A63 U728 U681 NP G61 U65 C730 C683 NP A86 A91 A762 A711 NP C87U92 C763 C712 NP G91 C96 U767 U716 NP U454 U448 C1104 C1187 NP C461 C455C1111 C1194 NP A463 A457 G1114 G1197 NP G469 G463 G1120 G1203 NP U470U464 C1122 NP NP A473 G467 C1125 U1206 NP C474 G468 G1126 C1207 NP G479G473 G1131 G1212 NP C480 G474 C1132 G1213 NP A485 A479 U1137 A1218 NPA486 A480 C1138 C1219 NP G487 G481 C1139 G1220 NP A489 A483 U1141 G1222NP C490 C484 C1142 C1223 NP C491 C485 C1143 G1224 NP A495 G489 U1147C1228 NP G496 C490 C1148 C1229 NP G499 G493 G1153 A1235 NP G512 G506G1166 G1248 NP C515 C509 G1169 G1251 NP C633 U576 G1284 G1365 NP G634G577 C1285 C1366 NP A635 G578 A1286 A1367 NP G636 G579 A1287 A1368 NPC637 U580 U1288 U1369 NP C638 C581 G1289 G1370 NP C764 C673 C1422 C1500NP G765 G674 G1423 G1501 NP A766 A675 A1424 A1502 NP G836 G745 G1492G1571 NP U837 NP* A1493 A1572 NP C838 NP C1494 C1573 NP C842 A749 C1498C1577 NP A843 A750 A1499 A1578 NP A846 A753 U1502 U1581 NP A882 A789A1538 A1617 NP U883 U790 U1539 U1618 NP C884 C791 C1540 C1619 NP G885A792 G1541 G1620 NP C889 C796 C1545 C1624 NP C890 G797 A1546 A1625 NPA1358 A1254 A2190 A2326 U350 C1365 C1261 U2197 U2333 A357 C1366 A1262G2198 G2334 G358 A1367 U1263 G2199 G2335 A359 U1368 A1264 U2200 U2336U360 A1369 A1265 A2201 A2337 A361 U1419 U1313 U2250 U2386 C407 C1423G1317 U2254 U2390 U411 U1432 U1326 C2263 C2399 U419 C1436 C1330 U2267U2403 U423 A1437 G1331 G2268 G2404 G424 G1438 G1332 A2269 A2405 U425G1443 G1337 G2274 G2410 C430 G1688 G1613 G2634 G2770 G679 A1691 A1616G2637 G2773 U682 C1692 C1617 C2638 C2774 A683 A1836 A1780 A3367 A3611A830 A1839 A1783 U3370 U3614 A833 U2052 U2011 C3583 C3827 U1004 G2053G2012 G3584 G3828 G1005 G2058 U2017 C3589 C3833 U1010 U2059 G2018 NP NPG1011 G2073 G2032 C3603 C3847 G1025 G2097 G2056 A3627 A3871 G1049 C2106C2065 C3636 C3880 C1058 U2107 C2066 U3637 U3881 U1059 G2284 G2251 G3917G4156 G1145 G2285 G2252 G3918 G4157 G1146 U2473 U2438 U4105 U4344 U1255C2475 C2440 C4107 C4346 C1257 C2476 U2441 C4108 C4347 C1258 A2479 G2444A4111 A4350 A1261 G2480 G2445 G4112 G4351 G1262 U2484 U2449 U4116 U4355U1266 G2489 G2454 G4121 G4360 G1271 U2528 U2493 U4160 U4399 U1310 G2529G2494 G4161 G4400 A1311 U2531 C2496 U4163 U4402 G1313 A2532 A2497 C4164C4403 A1314 C2534 C2499 U4166 U4405 C1316 U2535 U2500 U4167 U4406 U1317G2543 G2508 G4175 G4414 G1325 G2588 G2553 G4220 G4459 G1370 U2607 A2572A4239 A4478 A1389 U2610 C2575 U4242 U4481 U1392 A2612 A2577 A4244 A4483A1394 G2613 G2578 G4245 G4484 U1395 C2614 C2579 C4246 C4485 C1396 U2615U2580 U4247 U4486 U1397 G2617 G2582 G4249 G4488 A1399 A2622 A2587 A4254A4493 A1404 C2636 C2601 C4268 C4507 C1418 G2639 U2604 G4271 G4510 G1421G2642 G2607 A4274 A4513 G1424 G2643 G2608 G4275 G4514 G1425 U2648 U2613A4280 A4519 U1430 Protein L4 T56 T48 T62 T62 E67 P57 R49 S63 R63/S63^(n)S68 A58 A50 A64 A64 L69 R78 A68 H85 H85 P91 R79 R69 R86 R86 R92 V80 S70S87 S87 L96 Q82 NP Q89 Q89 Q98 A83 S72 G90 G90 V99 V84 I73 A91 A91 A100K85 K74 F92 F92 M101 Protein L22 R19 NP S20 S20 R72 R21 A10 L22 L22 I74Q22 R11 R23 R23 K75 V119 G79 A122 A122 G175 G120 P80 P123 P123 P176 S138K98 S141 S141 Q195 P139 R99 S142 S142 Q196 Protein L24 K81 N74 K89 K89Y95 R83 A76 N91 N91 Y97 G84 T77 G92 G92 I98 E85 G78 T93 T93 G99 ProteinL29 G37 G35 G38 G38 XX** G38 NP G39 G39 XX A39 Q36 A40 A40 XX P40 L37A41 A41 XX E41 Q38 S42 S42 XX P43 S40 L44 L44 XX Protein L37E G4 XX G4G4 XX T5 XX T5 T5 XX Protein L39E L14 XX K16 K16 K94 D15 XX Q17 Q17 A95N16 XX K18 K18 S96 Q17 XX Q19 Q19 Q97 W24 XX W26 W26 F111 W42 XX W44 W44L157 R43 XX R45 R45 R158 D46 XX K48 K48 R172 The residues in the 10-15 Åshell were determined to be those residues in the 50S ribosomal subunitthat are within 10-15 angstroms of the atoms of a model of a newlysynthesized peptide positioned in the center of the exit tunnel usingthe program MIDAS^(a). Conserved residues in 23S rRNA were determined bycomparison of sequences from the structure of H. marismortui 23SrRNA^(b) with # the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In cases of ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous # proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human mitochondria^(l). Sequence #alignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) with default parameters. *NP means that no homologousresidue has been identified. **XX means that no homologous protein hasbeen identified in that species. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession P12735 forprotein L4; GenBank accession R5HS22 for protein L22; GenBank accessionR5HS22 for protein L24; GenBank accession R5HS29 for protein L29;GenBank accession P32410 for protein L37E; GenBank accession P22452 forprotein L39E. ^(i)GenBank accession CAA26461 for protein L4; GenBankaccession CAA26465 for protein L22; GenBank accession R5EC24 for proteinL24; GenBank accession R5EC29 for protein L29; no GenBank accession for# protein L37E; no GenBank accession for protein L39E. ^(j)GenBankaccession JC4277 for protein L4; GenBank accession P24049 for proteinL22; GenBank accession P12749 for protein L24; GenBank accession R5RT35for protein L29; GenBank accession CAA47012 for protein L37E; GenBankaccession CAA57900 for protein L39E. ^(k)GenBank accessions P36578,NP_00959, S39803, and T09551 for protein L4; GenBank accession XP_057521for protein L22; GenBank accession AAA60279 and NP_000978 for proteinL24; GenBank accession AAA51648 for protein L29; GenBank accessionNP_000988 for protein L37E; GenBank accession NP_000991 for proteinL39E. ^(l)GenBank accession XP_049502 for protein L4; GenBank accessionXP_051279 for protein L22; GenBank accession XP_056380 for protein L24;no GenBank accession for protein L29; no GenBank accession for proteinL37E; GenBank accessions NP_059142 and NP_542984 for protein L39E.^(n)R63 is present in GenBank sequences T09551 and S39803 whereas S63 ispresent in GenBank sequences P36578 and NP_000959.

FIG. 30 shows a region of the large ribosomal subunit in which anantibiotic binds. FIG. 30(A) shows an enlarged portion of the largeribosomal subunit with the antibiotic tylosin bound at the top of thepolypeptide exit tunnel adjacent the peptidyl transferase site. FIGS.30(B) and 30(C) are views showing each half of a large ribosomal subunitcut along the polypeptide exit tunnel and are provided to orient thereader to show the tylosin binding site relative to the large ribosomalunit as a whole. FIG. 30(A) also shows two cavities defined by the wallof the polypeptide exit tunnel and are denoted as “cavity 1” and “cavity2.” In addition, FIG. 30(A) also shows a disaccharide binding pocket.The direction in which the newly synthesized polypeptide chains exitsthe ribosome through the polypeptide exit tunnel is denoted by an arrow.

Table 9 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define a first cavity within the wall ofpolypeptide exit tunnel (cavity 1). In addition, Table 9 identifieswhich of those residues that define corresponding residues from cavity 1in E. coli, Rattus, human, and human mitochondria. The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 9 Residues that Define Cavity 1 in the Ribosomal Peptide ExitTunnel Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C474 G468 G1126 C1207NP* A766 A675 A1424 A1502 NP A767 A676 A1425 A1503 NP U768 A677 A1426A1504 NP U883 U790 U1539 U1618 NP C884 C791 C1540 C1619 NP G885 A792G1541 G1620 NP A886 A793 A1542 A1621 NP U888 C795 C1544 C1623 NP C889C796 C1545 C1624 NP C890 G797 A1546 A1625 NP U1359 U1255 U2191 U2327U351 G1837 U1781 A3368 A3612 C831 A2100 A2059 A3630 A3874 A1052 A2101A2060 A3631 A3875 A1053 G2102 G2061 G3632 G3876 G1054 A2103 A2062 A3633A3877 A1055 C2475 C2440 C4107 C4346 C1257 C2476 U2441 C4108 C4347 C1258C2477 C2442 A4109 A4348 C1259 U2478 C2443 C4110 C4349 U1260 A2479 G2444A4111 A4350 A1261 A2538 A2503 A4170 A4409 A1320 Protein L4 P57 R49 S63R63/S63^(n) S68 A58 A50 A64 A64 L69 E59 E51 E65 E65 R70 S60 V52 S66 S66G71 F61 T53 W67 W67 F72 G62 G54 G68 G68 E73 S63 S55 T69 T69 Q74 G64 G56G70 G70 E75 R65 K57 R71 R71 R76 Q67 NP* V73 V73 L79 V70 P59 I76 I76 L82P71 W60 P77 P77 H83 K72 R61 R78 R78 P84 L73 Q62 V79 V79 D85 D74 G64 G81G81 F87 G75 T65 G82 G82 A88 R76 G66 G83 G83 T89 Cavity residues wereidentified using the program MIDAS^(a). Conserved residues in 23S rRNAwere determined by comparison of sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). In cases # ofribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the corresponding #sequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA). *NP means that nohomologous residue has been identified. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge. (1988). “The MIDAS Display System,” J.Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619^(c)GenBank accession J01695 ^(d)GenBank accession 2624399 ^(e)GenBankaccession M11167 ^(f)GenBank accession 13683 ^(g)GenBank accessionP12735 for protein L4. ^(i)GenBank accession CAA26461 for protein L4.^(j)GenBank accession JC4277 for protein L4. ^(k)GenBank accessionsP36578, NP_00959, S39803, and T09551 for protein L4. ^(l)GenBankaccession XP_049502 for protein L4. ^(n)R63 is present in GenBanksequences T09551 and S39803 whereas S63 is present in GenBank sequencesP36578 and NP_000959.

Table 10 identifies the residues in the H. marismortui 50S ribosomalsubunit that together define a second cavity in the wall of polypeptideexit tunnel (cavity 2). In addition, Table 10 identifies which of thoseresidues that define corresponding residues from cavity 2 in E. coli,Rattus, human, and human mitochondria. The non-conserved residues wereidentified as described previously with respect to Tables 5A and 5B.

TABLE 10 Residues that Define Cavity 2 in the Ribosomal Peptide ExitTunnel Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA U831 C740 U1488 U1567NP* U832 U741 C1489 C1568 NP G833 A742 C1490 C1569 NP G834 A743 NP NP NPU835 U744 U1491 U1570 NP G836 G745 G1492 G1571 NP U837 NP A1493 A1572 NPC838 NP C1494 C1573 NP C839 U746 G1495 G1574 NP U840 U747 U1496 U1575 NPA841 G748 G1497 G1576 NP A843 A750 A1499 A1578 NP A844 A751 A1500 A1579NP U845 A752 A1501 A1580 NP A846 A753 U1502 U1581 NP C847 U754 C1503C1582 NP C848 U755 G1504 G1583 NP C849 A756 G1505 G1584 NP C1753 C1675C2695 C2833 NP A1754 A1676 A2696 A2834 U734 G1837 U1781 A3368 A3612 C831U1838 U1782 U3369 U3613 C832 A1839 A1783 U3370 U3614 A833 G2099 A2058G3629 G3873 G1051 A2100 A2059 A3630 A3874 A1052 A2103 A2062 A3633 A3877A1055 U2615 U2580 U4247 U4486 U1397 G2616 G2581 G4248 G4487 G1398 U2621U2586 U4253 U4492 C1403 A2622 A2587 A4254 A4493 A1404 G2643 G2608 G4275G4514 G1425 C2644 U2609 U4276 U4515 U1426 U2645 C2610 U4277 U4516 U1427G2646 C2611 U4278 U4517 U1428 C2647 C2612 U4279 U4518 C1429 Cavityresidues were determined using the program MIDAS^(a). Conserved residuesin 23S rRNA were determined by comparison of sequences from thestructure of H. marismortui 23S rRNA^(b) with the correspondingsequences of aligned genomic DNA encoding the homologous 23S rRNA (E.coli ^(c), Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)).Sequence alignments # were determined with the program MegAlign(DNASTAR, Madison, Wisconsin, USA) using default parameters. *NP meansthat no homologous residue has been identified. ^(a)T. E. Ferrin, C. C.Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDAS Display System”J. Mol. Graphics, 6(1): 13-27, 36-37. ^(b)GenBank accession AF034619^(c)GenBank accession J01695 ^(d)GenBank accession 2624399 ^(e)GenBankaccession M11167 ^(f)GenBank accession 13683

Tables 9 and 10, however, define only two of many cavities disposedwithin the wall of the polypeptide exit tunnel. However, by using theatomic co-ordinates and molecular modeling methodologies describedherein, the skilled artisan may identify the residues (contributed byamino acids, nucleotides or a combination of both) that together defineother cavities within the wall of the polypeptide exit tunnel.

In addition, by using the atomic co-ordinates described herein, theskilled artisan can identify the antibiotic binding site of anyantibiotic of interest. This information also provides contact sitesbetween an antibiotic and the residues in a ribosome or ribosomalsubunit, which can be used to advantage in the design of novel ormodified protein synthesis inhibitors. The binding or contact sites fora variety of antibiotics are discussed in more detail below.

Table 11A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of an anisomycin bindingpocket (5.8 Å shell). In addition, Table 11A identifies thecorresponding residues that define at least a portion of the anisomycinbinding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Table 11B identifies the residues in the H. marismortui 50Sribosomal subunit that together define a broader portion of ananisomycin binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 11A Residues that Define the Anisomycin Binding Pocket (5.8 Åshell) Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA A2100^(v) A2059 A3630A3874 A1052 G2102^(v) G2061 G3632 G3876 G1054 A2103^(v) A2062 A3633A3877 A1055 G2482^(v) G2447 G4114 G4353 G1264 A2486^(v) A2451 A4118A4357 A1268 C2487^(h,v) C2452 C4119 C4358 C1269 A2488^(v) A2453 U4120U4359 U1270 U2535^(h,v) U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407C1318 A2538^(v) A2503 A4170 A4409 A1320 U2539^(h,v) U2504 U4171 U4410U1321 G2540^(v) G2505 G4172 G4411 G1322 U2541^(v) U2506 U4173 U4412U1323 U2620^(v) U2585 U4252 U4991 U1402 The residues that define thebinding pocket (5.8 Å shell) were determined as those residues in the50S ribosomal subunit that are located within 5.8 angstroms of # theatoms of anisomysin using the program MIDAS^(a). Conserved residues in23S rRNA were determined by comparison of sequences from the structureof H. marismortui 23S rRNA^(b) with the corresponding sequences ofaligned genomic DNA encoding the homologous 23S rRNA (E. coli ^(c),Rattus norvegicus ^(d), Human^(e) or Human # mitochondria^(f)). Sequencealignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) with default parameters. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. J. Comput. Chem. (1993) 14:195-205.^(v)Residues in van der Waals contact with the antibiotic are defined asthose residues within rmin of any atom in the antibiotic, where rmin =21/6s and is the minimum in the Lennard-Jones pair potential (seeComputer Simulation of Liquids; Alien, M. P. and Tildesley, D. J.;Oxford University Press: New York, 1992, 11.). The Lennard-Jones ss aretaken from the OPLS-AA force # field (see, Jorgensen, W. L.; Maxwell, D.S. and Tirado-Rives, J. J. Am. Chem. Soc, 1996, 118, 11225-11236 and #Jorgensen, W. L. BOSS, Version 4.3; Yale University: New Haven, CT,2002).

TABLE 11B Residues that Define the Anisomycin Binding Pocket (5.8 Å-12.6Å shell) Corre- Corre- sponding sponding Corre- Corre- Residue inResidue sponding sponding Human H. marismortui in E. Residue in Residuein Mitochon- Residue coli Rattus Human dria 23S rRNA A631 A574 G1282G1363 NP* A632 A575 C1283 C1364 NP C633 U576 G1284 G1365 NP G2073 G2032C3603 C3847 G1025 A2095 A2054 G3625 G3869 A1047 A2096 C2055 A3626 A3870C1048 G2097 G2056 A3627 A3871 G1049 C2098 G2057 A3628 A3872 A1050 G2099A2058 G3629 G3873 G1051 G2102 G2061 G3632 G3876 G1054 C2104 C2063 C3634C3878 C1056 C2105 C2064 C3635 C3879 C1057 A2474 A2439 A4106 A4345 A1256G2481 G2446 G4113 G4352 G1263 U2484 U2449 U4116 U4355 U1266 A2485 A2450A4117 A4356 A1267 G2489 G2454 G4121 G4360 G1271 A2490 G2455 G4122 G4361C1272 A2532 A2497 C4164 C4403 A1314 C2533 C2498 C4165 C4404 C1315 C2534C2499 U4166 U4405 C1316 G2537 G2502 G4169 G4408 G1319 C2542 C2507 C4174C4413 U1324 G2543 G2508 G4175 G4414 G1325 G2588 G2553 G4220 G4459 G1370U2607 A2572 A4239 A4478 A1389 C2608 C2573 C4240 C4479 C1390 G2609 G2574G4241 G4480 G1391 U2610 C2575 U4242 U4481 U1392 C2611 G2576 G4243 G4482G1393 A2612 A2577 A4244 A4483 A1394 G2618 G2583 G4250 G4489 G1400 U2619U2584 U4251 U4490 U1401 A2637 A2602 A4269 A4508 A1419 G2646 C2611 U4278U4517 U1428 L3 W242 Q150 W257 W257 G246 The residues that define thebinding pocket (5.8 Å-12.6 Å shell) were determined as those residues inthe 50S ribosomal subunit that are located within 5.8-12.6 angstroms ofthe atoms of anisomysin using the program MIDAS^(a). Conserved residueswere determined by comparison between the proposed secondary structuresof H. marismortui ^(b), E. coli ^(b), and Rattus norvegicus ^(c),Human^(d), and # Human Mitochondria^(e). Sequence alignments weredetermined with the program MegAlign (DNASTAR, Madison, Wisconsin, USA)using defaul parameters. *NP means that no homologous residue has beenidentified. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R.Langridge (1988) “The MIDAS Display System” J. Mol. Graphics,6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBank accessionJ01695 ^(d)GenBank accession 2624399 ^(e)GenBank accession M11167^(f)GenBank accession 13683 ^(g)GenBank accession AAA86859 for proteinL3. ^(i)GenBank accession CAA26460 for protein L3. ^(j)GenBank accessionP21531 for protein L3. ^(k)GenBank accession NP_000964 for protein L3.^(l)GenBank accession P09001 for protein L3.

Table 12A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a blasticidin bindingpocket (5.8 Å shell). In addition, Table 12A identifies thecorresponding residues that define at least a portion of the blasticidinbinding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Table 12B identifies the residues in the H. marismortui 50Sribosomal subunit that together define a broader portion of ablasticidin binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 12A Residues that Define the Blasticidin Binding Pocket (5.8 Åshell) Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA A2007^(v) A1966 A3538A3782 A959 C2104^(v) C2063 C3634 C3878 C1056 C2105^(h,v) C2064 C3635C3879 C1057 C2106^(v) C2065 C3636 C3880 C1058 G2284^(h,v) G2251 G3917G4156 G1145 G2285^(h,v) G2252 G3918 G4157 G1146 G2286^(h,v) G2253 G3919G4158 G1147 C2287^(v) C2254 C3920 C4159 C1148 U2473^(v) U2438 U4105U4344 U1255 A2474^(h,v) A2439 A4106 A4345 A1256 A2485^(v) A2450 A4117A4356 A1267 A2486^(v) A2451 A4118 A4357 A1268 U2620^(v) U2585 U4252U4491 U1402 G2627^(v) G2592 G4259 G4498 G1409 U2628^(v) U2593 U4260U4499 G1410 C2629^(v) C2594 C4261 C4500 A1411 G2634^(v) G2599 G4266G4505 U1416 A2635^(h,v) A2600 A4267 A4506 C1417 C2636^(h,v) C2601 C4268C4507 C1418 A2637^(v) A2602 A4269 A4508 A1419 The residues that definethe binding pocket (5.8 Å shell) were determined as those residues inthe 50S ribosomal subunit that are located within 5.8 angstroms of theatoms of Blasticidin using the program MIDAS^(a). Conserved residues in23S rRNA were determined by comparison of sequences from the structureof H. marismortui 23S rRNA^(b) with the corresponding sequences ofaligned genomic DNA encoding the # homologous 23S rRNA (E. coli ^(c),Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)). Sequencealignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(v)Residues in van der Waals contact with the antibiotic are defined asthose residues within rmin of any atom in the antibiotic, where rmin =21/6s and is the minimum in the Lennard-Jones pair potential (seeComputer Simulation of Liquids; Alien, M. P. and Tildesley, D. J.;Oxford University Press: New York, 1992, 11.). The Lennard-Jones ss aretaken from the OPLS-AA force field (see, Jorgensen, W. L.; Maxwell, D.S. and # Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118, 11225-11236 andJorgensen, W. L. BOSS, Version 4.3; Yale University: New Haven, CT,2002).

TABLE 12B Residues that Define the Blasticidm Binding Pocket (5.8 Å-12.6Å shell) Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA G885 A792 G1541 G1620NP* U1980 U1939 U3511 U3755 U932 A1981 U1940 G3512 G3756 C933 C1982C1941 C3513 C3757 A934 C2006 C1965 C3537 C3781 U958 U2008 C1967 U3539U3783 U960 G2102 G2061 G3632 G3876 G1054 A2103 A2062 A3633 A3877 A1055U2107 C2066 U3637 U3881 U1059 G2110 G2069 U3640 U3884 G1062 G2111 A2070G3641 G3885 NP A2112 A2071 A3642 A3886 NP G2113 C2072 G3643 G3887 NPC2114 C2073 C3644 C3888 NP U2282 U2249 U3915 U4154 U1143 G2283 G2250G3916 G4155 G1144 G2288 G2255 G3921 G4160 G1149 G2289 G2256 G3922 G4161A1150 G2471 G2436 G4103 G4342 G1253 C2472 G2437 U4104 U4343 U1254 C2475C2440 C4107 C4346 C1257 C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109A4348 C1259 U2478 C2443 C4110 C4349 U1260 G2481 G2446 G4113 G4352 G1263G2482 G2447 G4114 G4353 G1264 U2484 U2449 U4116 U4355 U1266 C2487 C2452C4119 C4358 C1269 G2529 G2494 G4161 G4400 A1311 C2530 G2495 A4162 A4401C1312 U2531 C2496 U4163 U4402 G1313 A2532 A2497 C4164 C4403 A1314 C2536C2501 C4168 C4407 C1318 U2619 U2584 U4251 U4490 U1401 U2621 U2586 U4253U4492 C1403 A2622 A2587 A4254 A4493 A1404 G2623 G2588 G4255 G4494 G1405A2624 A2589 A4256 A4495 A1406 C2625 A2590 C4257 C4496 C1407 G2630 G2595G4262 G4501 G1412 A2633 A2598 A4265 A4504 A1415 G2638 G2603 G4270 G4509G1420 G2639 U2604 G4271 G4510 G1421 U2640 U2605 U4272 U4511 U1422 G2642G2607 A4274 A4513 G1424 G2643 G2608 G4275 G4514 G1425 Protein L2 G204G235 G214 G214 W281 G205 E236 N215 N215 A282 R206 G237 H216 H216 G283Protein L10E D109 XX** T113 T113 XX G110 XX G114 G114 XX M111 XX M115M115 XX R112 XX R116 R116 XX The residues that define the binding pocket(5.8 Å-12.6 Å shell) were determined as those residues in the 50Sribosomal subunit that are located within 5.8-12.6 angstroms of theatoms of blasticidin using the program MIDAS^(a). Conserved residues in23S rRNA were determined by # comparison of sequences from the structureof H. marismortui 23S rRNA^(b) with the corresponding sequences ofaligned # genomic DNA encoding the homologous 23S rRNA (E. coli ^(c),Rattus norvegicus ^(d), Human^(e) or Human mitochondria^(f)). In casesof ribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins # inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Humanmitochondria^(l). Sequence alignments were # determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.*NP means that no homologous residue has been identified. **XX meansthat no homologous protein has been identified in that species. ^(a)T.E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDASDisplay System” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBankaccession AF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession 13683^(g)GenBank accession R5HS2L for protein L2; GenBank accession 15825950for protein L10E. ^(i)GenBank accession CAA26463 for protein L2; NOGenBank accession for protein L10E. ^(j)GenBank accession R5RTL8 forprotein L2; GenBank accession NP_112362 for protein L10E. ^(k)GenBankaccession NP_000964 for protein L2; GenBank accession NP_006004 forprotein L10E. ^(l)GenBank accession XP_004140 for protein L2; no GenBankaccession for protein L10E.

Table 13A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a carbomycin Abinding pocket (5.8 Å shell). In addition, Table 13A identifies thecorresponding residues that define at least a portion of the carbomycinA binding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Further, Table 13B identifies the residues in the H.marismortui 50S ribosomal subunit that together define a broader portionof a carbomycin A binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 13A Residues that Define the Carbomycin A Binding Pocket (5.8 Åshell) Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C839^(v) U746 G1495G1574 NP* C2098^(v) G2057 A3628 A3872 A1050 G2099^(h,v) A2058 G3629G3873 G1051 A2100^(v) A2059 A3630 A3874 A1052 G2102^(h,v) G2061 G3632G3876 G1054 A2103^(h,v) A2062 A3633 A3877 A1055 C2104 C2063 C3634 C3878C1056 A2486^(v) A2451 A4118 A4357 A1268 C2487^(v) C2452 C4119 C4358C1269 A2538^(h,v) A2503 A4170 A4409 A1320 U2539^(h,v) U2504 U4171 U4410U1321 G2540^(h,v) G2505 G4172 G4411 G1322 U2541 U2506 U4173 U4412 U1323U2620^(v) U2585 U4252 U4491 U1402 C2644^(v) U2609 U4276 U4515 A1426G2646^(v) C2611 U4278 U4517 U1428 Protein L22 M130^(v) K90 H133 H133V187 The residues that define the binding pocket (5.8 Å shell) weredetermined as those residues in the 50S ribosomal subunit that arelocated within 5.8 angstroms of the atoms of carbomycin A using theprogram MIDAS ^(a) . Conserved residues in 23S rRNA were determined bycomparison # of sequences from the structure of H. marismortui 23SrRNA^(b) with the # corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human # mitochondria^(f)). In cases of ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human # mitochondria^(l). Sequencealignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) with default parameters. *NP means that no homologousresidue has been identified. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession R5HS22 forprotein L22. ^(h)Indicates residue that is within hydrogen-bondingdistance (3.5 Å) of the ligand; for example, see Jorgensen, W L, Nguyen,T B. (1993) J. Comput. Chem. 14:195-205. ^(i)GenBank accession CAA26465for protein L22. ^(j)GenBank accession P24049 for protein L22.^(k)GenBank accession XP_057521 for protein L22. ^(l)GenBank accessionXP_051279 for protein L22. ^(m)This residue is actually at 5.85 Å.^(v)Residues in van der Waals contact with the antibiotic are defined asthose residues within rmin of any atom in the antibiotic, where rmin =21/6s and is the minimum in the Lennard-Jones pair potential (seeComputer Simulation of Liquids; Alien, M. P. and Tildesley, D. J.;Oxford # University Press: New York, 1992, 11.). The Lennard-Jones ssare taken from the OPLS-AA force field (see, Jorgensen, W. L.; Maxwell,D. S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118, 11225-11236 andJorgensen, W. L. BOSS, Version 4.3; Yale # University: New Haven, CT,2002).

TABLE 13B Residues that Further Define the Carbomycin A Binding PocketCorre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C633 U576 G1284 G1365NP* U835 U744 U1491 U1570 NP G836 G745 G1492 G1571 NP U837 NP A1493A1572 NP C838 NP C1494 C1573 NP U840 U747 U1496 U1575 NP A841 G748 G1497G1576 NP A843 A750 A1499 A1578 NP A844 A751 A1500 A1579 NP U845 A752A1501 A1580 NP A846 A753 U1502 U1581 NP A882 A789 A1538 A1617 NP U883U790 U1539 U1618 NP U1359 U1255 U2192 U2327 U351 G1837 U1781 A3368 A3612C831 U1838 U1782 U3369 U3613 C832 C2056 A2015 A3587 A3831 A1008 U2057U2016 C3588 C3832 U1009 G2073 G2032 C3603 C3847 G1025 A2095 A2054 G3625G3869 A1047 A2096 C2055 A3626 A3870 C1048 G2097 G2056 A3627 A3871 G1049A2101 A2060 A3631 A3875 A1053 C2105 C2064 C3635 C3879 C1057 A2474 A2439A4106 A4345 A1256 C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109 A4348C1259 U2478 C2443 C4110 C4349 U1260 G2481 G2446 G4113 G4352 G1263 G2482G2447 G4114 G4353 G1264 A2485 A2450 A4117 A4356 A1267 A2488 A2453 U4120U4359 A1270 G2489 G2454 G4121 G4360 G1271 U2534 C2499 U4166 U4405 C1316U2535 U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407 C1318 G2537 G2502G4169 G4408 G1319 C2542 C2507 C4174 C4413 U1324 G2543 G2508 G4175 G4414G1325 U2607 A2572 A4239 A4478 A1389 C2608 C2573 C4240 C4479 C1390 U2610C2575 U4242 U4481 U1392 G2611 G2576 G4243 G4482 G1393 A2612 A2577 A4244A4483 A1394 C2614 C2579 C4246 C4485 C1396 U2615 U2580 U4247 U4486 U1397G2616 G2581 G4248 G4487 G1398 G2618 G2583 G4250 G4489 G1400 U2619 U2584U4251 U4490 U1401 U2621 U2586 U4253 U4492 C1403 A2622 A2587 A4254 A4493A1404 A2637 A2602 A4269 A4508 A1419 G2643 G2608 G4275 G4514 G1425 U2645C2610 U4277 U4516 U1427 C2647 C2612 U4279 U4518 U1429 L3 W242 Q150 W257W257 G246 L4 F61 T53 W67 W67 F72 G62 G54 G68 G68 E73 S63 S55 T69 T69 Q74G64 G56 G70 G70 E75 R65 K57 R71 R71 R76 G66 NP A72 A72 G78 Q67 NP V73V73 L79 A129 A89 A132 A132 A186 G131 G91 G134 G134 A188 R132 R92 R135R135 H189 The residues that define the binding pocket (5.8 Å-12.6 Åshell) were determined as those residues in the 50S ribosomal subunitthat are located within 5.8-12.6 angstroms of the atoms of carbomycin Ausing the program MIDAS^(a). Conserved residues in 23S rRNA weredetermined # by comparison of sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human # mitochondria^(f)). In cases ofribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Human #mitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.*NP means that no homologous residue has been identified. ^(a)T. E.Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDASDisplay System” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBankaccession AF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession 13683^(g)GenBank accession AAA86859 for protein L3; GenBank accession P12735for protein L4. ^(i)GenBank accession CAA26460 for protein L3; GenBankaccession CAA26461 for protein L4. ^(j)GenBank accession P21531 forprotein L3; GenBank accession JC4277 for protein L4. ^(k)GenBankaccession NP_000958 for protein L3; GenBank accessions P36578,NP_000959, S39803, and T09551 for protein L4. ^(l)GenBank accessionP09001 for protein L3; GenBank accession XP_049502 for protein L4.

Table 14A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a tylosin bindingpocket (5.8 Å shell). In addition, Table 14A identifies thecorresponding residues that define at least a portion of the tylosinbinding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Further, Table 14B identifies the residues in the H.marismortui 50S ribosomal subunit that together define a broader portionof a tylosin binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 14A Residues that Define the Tylosin Binding Pocket (5.8 Å shell)Corre- sponding Corre- Corre- Residue in Corresponding sponding spondingHuman H. marismortui Residue in E. Residue in Residue in Mitochon-Residue coli Rattus Human dria 23S rRNA C839^(v) U746 G1495 G1574 NP*A841^(h,v) G748 G1497 G1576 NP A843^(v) A750 A1499 A1578 NP A844^(h,v)A751 A1500 A1579 NP U845^(v) A752 A1501 A1580 NP A846^(v) A753 U1502U1581 NP G1837 U1781 A3368 A3612 C831 C2098^(v) G2057 A3628 A3872 A1050G2099^(h,v) A2058 G3629 G3873 G1051 A2100^(v) A2059 A3630 A3874 A1052G2102^(v) G2061 G3632 G3876 G1054 A2103^(h,v) A2062 A3633 A3877 A1055A2486^(v) A2451 A4118 A4357 A1268 A2538^(h,v) A2503 A4170 A4409 A1320U2539^(v) U2504 U4171 U4410 U1321 G2540^(v) G2505 G4172 G4411 G1322U2541 U2506 U4173 U4412 U1323 U2620^(m,,v) U2585 U4252 U4491 U1402C2644^(v) U2609 U4276 U4515 U1426 U2645^(v) C2610 U4277 U4516 U1427G2646^(v) C2611 U4278 U4517 U1428 Protein L22 M130^(v) K90 H133 H133V187 The residues that define the binding pocket (5.8 Å shell) weredetermined as those residues in the 50S ribosomal subunit that arelocated within 5.8 angstroms of the atoms of tylosin using the programMIDAS^(a). Conserved residues in 23S rRNA were determined by comparisonof sequences from the # structure of H. marismortui 23S rRNA^(b) withthe corresponding sequences of aligned genomic DNA encoding thehomologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d), Human^(e) orHuman mitochondria^(f)). In cases of # ribosomal proteins, comparisonswere also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human mitochondria^(l). Sequence #alignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. *NP means that no homologousresidue has been identified. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1): 13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession R5HS22 forprotein L22. ^(h)Indicates residue that is within hydrogen-bondingdistance (3.5 Å) of the ligand; for example, see Jorgensen, W L, Nguyen,T B. (1993) J. Comput. Chem. 14:195-205. ^(i)GenBank accession CAA26465for protein L22. ^(j)GenBank accession P24049 for protein L22.^(k)GenBank accession XP_057521 for protein L22. ^(l)GenBank accessionXP_051279 for protein L22. ^(m)This residue is actually at 5.87 Å.^(v)Residues in van der Waals contact with the antibiotic are defined asthose residues within rmin of any atom in the antibiotic, where rmin =21/6 s and is the minimum in the Lennard-Jones pair potential (seeComputer Simulation of Liquids; Alien, M. P. and Tildesley, D. J.;Oxford # University Press: New York, 1992, 11.). The Lennard-Jones ssare taken from the OPLS-AA force field (see, Jorgensen, W. L.; Maxwell,D. S. and Tirado-Rives, J. J. Am. Chem. Soc, 1996, 118, 11225-11236 andJorgensen, W. L. BOSS, Version 4.3; Yale University: New Haven, CT,2002).

TABLE 14B Residues that Define the Tylosin Binding Pocket (5.8 Å-12.6 Åshell) Corre- sponding Corre- Corre- Residue in Corresponding spondingsponding Human H. marismortui Residue in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C633 U576 G1284 G1365NP* U835 U744 U1491 U1570 NP G836 G745 G1492 G1571 NP U837 NP A1493A1572 NP C838 NP C1494 C1573 NP U840 U747 U1496 U1575 NP C842 A749 C1498C1577 NP C847 U754 C1503 C1582 NP A882 A789 A1538 A1617 NP U883 U790U1539 U1618 NP U1359 U1255 U2191 U2327 U351 G1688 G1613 G2634 G2770 G679A1689 A1614 C2635 C2771 A680 C1690 C1615 A2636 A2772 U681 C1692 C1617C2638 C2774 A683 G1694 G1619 G2640 G2776 A685 A1836 A1780 A3367 A3611A830 C2056 A2015 A3587 A3831 A1008 U2057 U2016 C3588 C3832 U1009 G2073G2032 C3603 C3847 G1025 A2095 A2054 G3625 G3869 A1047 A2096 C2055 A3626A3870 C1048 G2097 G2056 A3627 A3871 G1049 A2101 A2060 A3631 A3875 A1053C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879 C1057 A2474 A2439A4106 A4345 A1256 C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109 A4348C1259 U2478 C2443 C4110 C4349 U1260 G2481 G2446 G4113 G4352 G1263 G2482G2447 G4114 G4353 G1264 A2485 A2450 A4117 A4356 A1267 C2487 C2452 C4119C4358 C1269 A2488 A2453 U4120 U4359 A1270 U2535 U2500 U4167 U4406 U1317C2536 C2501 C4168 C4407 C1318 G2537 G2502 G4169 G4408 G1319 C2542 C2507C4174 C4413 U1324 U2607 A2572 A4239 A4478 A1389 C2608 C2573 C4240 C4479C1390 U2610 C2575 U4242 U4481 U1392 G2611 G2576 G4243 G4482 G1393 A2612A2577 A4244 A4483 A1394 C2614 C2579 C4246 C4485 C1396 U2615 U2580 U4247U4486 U1397 G2616 G2581 G4248 G4487 G1398 G2618 G2583 G4250 G4489 G1400U2619 U2584 U4251 U4490 U1401 U2621 U2586 U4253 U4492 C1403 A2622 A2587A4254 A4493 A1404 G2643 G2608 G4275 G4514 G1425 C2647 C2612 U4279 U4518C1429 U2648 U2613 A4280 A4519 U1430 Protein L3 W242 Q150 W257 W257 G246Protein L4 G62 G54 G68 G68 E73 S63 S55 T69 T69 Q74 G64 G56 G70 G70 E75R65 K57 R71 R71 R76 G66 NP A72 A72 G78 Q67 NP V73 V73 L79 Protein L22R128 R88 R131 R131 K184 A129 A89 A132 A132 A186 G131 G91 G134 G134 A188R132 R92 R135 R135 H189 Protein L37E T1 XX** M1 M1 XX G2 XX T2 T2 XX A3XX K3 K3 XX G4 XX G4 G4 XX T5 XX T5 T5 XX P6 XX S6 S6 XX S7 XX S7 S7 XXQ8 XX F8 F8 XX The residues that define the binding pocket (5.8 Å-12.6 Åshell) were determined as those residues in the 50S ribosomal subunitthat are located within 5.8-12.6 angstroms of the atoms of tylosin usingthe program MIDAS^(a). Conserved residues in 23S rRNA were determined by# comparison of sequences from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In cases # of ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human # mitochondria^(l). Sequencealignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) with default parameters. *NP means that no homologousresidue has been identified. ** XX means that no homologous protein hasbeen identified in that species. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession AAA86859 forprotein L3; GenBank accession P12735 for protein L4; GenBank accessionR5HS24 for protein L22; GenBank accession P32410 for protein L37E.^(i)GenBank accession CAA26460 for protein L3; GenBank accessionCAA26461 for protein L4; GenBank accession CAA26465 for protein L22; noGenBank accession for protein L37E. ^(j)GenBank accession P21531 forprotein L3; GenBank accession JC4277 for protein L4; GenBank accessionP24049 for protein L22; GenBank accession CAA47102 for protein L37E.^(k)GenBank accession NP_000958 for protein L3; GenBank accessionsP36578, NP_000959, S39803, and T09551 for protein L4; GenBank accessionXP_051279 for protein L22; GenBank accession NP_000988 for protein L37E.^(l)GenBank accession P09001 for protein L3; GenBank accession XP_049502for protein L4; GenBank accession XP_051279 for protein L22; NO GenBankaccession for protein L37E.

Table 15A identifies the residues in the H marismortui 50S ribosomalsubunit that together define at least a portion of a sparsomycin bindingpocket (5.8 Å shell). In addition, Table 15A identifies thecorresponding residues that define at least a portion of the sparsomycinbinding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Further, Table 15B identifies the residues in the H.marismortui 50S ribosomal subunit that together define a broader portionof a sparsomycin binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 15A Residues that Define the Sparsomycin Binding Pocket (5.8 Åshell) Corre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA A2486^(h,v) A2451A4118 A4357 A1268 C2487^(v) C2452 C4119 C4358 C1269 A2488^(m,v) A2453U4120 U4359 A1270 G2540^(m,v) G2505 G4172 G4411 G1322 U2541^(v) U2506U4173 U4412 U1323 C2542 C2507 C4174 C4413 U1324 C2608^(v) C2573 C4240C4479 C1390 U2619^(h,v) U2584 U4251 U4490 U1401 U2620^(h,v) U2585 U4252U4491 U1402 C2636 C2601 C4268 C4507 C1418 A2637^(h,v) A2602 A4269 A4508A1419 The residues that define the binding pocket (5.8 Å shell) weredetermined as those residues in the 50S ribosomal subunit that arelocated within 5.8 angstroms of the atoms of sparsomycin using theprogram MIDAS^(a). Conserved residues in 23S rRNA were determined bycomparison of # sequences from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). Sequence alignments were #determined with the program MegAlign (DNASTAR, Madison, Wisconsin, USA)using default parameters. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis,and R. Langridge (1988) “The MIDAS Display System” J. Mol. Graphics,6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBank accessionJ01695 ^(d)GenBank accession 2624399 ^(e)GenBank accession M11167^(f)GenBank accession 13683 ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(m)Residue A2488 is actually 6.0 Å; residue G2540 is actually 5.85 Å.^(v)Residues in van der Waals contact with the antibiotic are defined asthose residues within rmin of any atom in the antibiotic, where rmin =21/6s and is the minimum in the Lennard-Jones pair potential (seeComputer Simulation of Liquids; Alien, M. P. and Tildesley, D. J.;Oxford # University Press: New York, 1992,11.). The Lennard-Jones ss aretaken from the OPLS-AA force field (see, Jorgensen, W. L.; Maxwell, D.S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118, 11225-11236 andJorgensen, W. L. BOSS, Version 4.3; Yale University: New Haven, CT,2002).

TABLE 15B Residues that Define the Sparsomycin Binding Pocket (5.8Å-12.6 Å shell) Corre- Corre- sponding sponding Corre- Corre- Residue inResidue sponding sponding Human H. marismortui in E. Residue in Residuein Mitochon- Residue coli Rattus Human dria 23S rRNA G2073 G2032 C3603C3847 G1025 G2102 G2061 G3632 G3876 G1054 A2103 A2062 A3633 A3877 A1055C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879 C1057 C2106 C2065C3636 C3880 C1058 G2284 G2251 G3917 G4156 G1145 G2285 G2252 G3918 G4157G1146 G2286 G2253 G3919 G4158 G1147 U2473 U2438 U4105 U4344 U1255 A2474A2439 A4106 A4345 A1256 G2482 G2447 G4114 G4353 G1264 A2485 A2450 A4117A4356 A1267 G2498 C2463 C4130 C4369 U1280 U2527 U2492 U4159 U4398 U1309U2528 U2493 U4160 U4399 U1310 G2529 G2494 G4161 G4400 A1311 C2530 G2495A4162 A4401 C1312 A2532 A2497 C4164 C4403 A1314 C2534 C2499 U4166 U4405C1316 U2535 U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407 C1318 A2538A2503 A4170 A4409 A1320 U2539 U2504 U4171 U4410 U1321 G2543 G2508 G4175G4414 G1325 G2588 G2553 G4220 G4459 G1370 U2589 U2554 U4221 U4460 U1371U2590 U2555 U4222 U4461 U1372 U2607 A2572 A4239 A4478 A1389 G2609 G2574G4241 G4480 G1391 U2610 C2575 U4242 U4481 U1392 G2611 G2576 G4243 G4482G1393 G2617 G2582 G4249 G4488 A1399 G2618 G2583 G4250 G4489 G1400 U2621U2586 U4253 U4492 C1403 A2622 A2587 A4254 A4493 A1404 G2627 G2592 G4259G4498 G1409 A2635 A2600 A4267 A4506 C1417 G2638 G2603 G4270 G4509 G1420G2639 U2604 G4271 G4510 G1421 U2640 U2605 U4272 U4511 U1422 G2643 G2608G4275 G4514 G1425 Protein L3 W242 Q150 W257 W257 G246 The residues thatdefine the binding pocket (5.8 A-12.6 Å shell) were determined as thoseresidues in the 50S ribosomal subunit that are located within 5.8-12.6angstroms of the atoms of sparsomycin using the program MIDAS^(a).Conserved residues in 23S rRNA were determined # by comparison ofsequences from the structure of H. marismortui 23S rKNA^(b) with thecorresponding sequences of aligned genomic DNA encoding the homologous23S rRNA (E. coli ^(c), Rattus norvegicus ^(d), Human^(e) or Humanmitochondria^(f)). In cases # of ribosomal proteins, comparisons werealso made between the H. marismortui structural protein sequence^(g) andthe corresponding sequences of aligned protein sequences encodinghomologous proteins in E. coli ^(i), Rattus norvegicus ^(j), Human^(k)or Human mitochondria^(l). Sequence alignments were determined with theprogram MegAlign (DNASTAR, Madison, Wisconsin, USA) with defaultparameters. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R.Langridge (1988) “The MIDAS Display System” J. Mol. Graphics,6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBank accessionJ01695 ^(d)GenBank accession 2624399 ^(e)GenBank accession M11167^(f)GenBank accession 13683 ^(g)GenBank accession AAA86859 for proteinL3. ^(i)GenBank accession CAA26460 for protein L3. ^(j)GenBank accessionP21531 for protein L3. ^(k)GenBank accession NP_000958 for protein L3.^(l)GenBank accession P09001 for protein L3.

Table 16A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a virginiamycin Mbinding pocket (5.8 Å shell). In addition, Table 16A identifies thecorresponding residues that define at least a portion of thevirginiamycin M binding pocket in E. coli, Rattus, human, and humanmitochondria large subunit. Further, Table 16B identifies the residuesin the H. marismortui 50S ribosomal subunit that together define abroader portion of a virginiamycin M binding pocket (5.8 Å-12.6 Åshell). The non-conserved residues were identified as describedpreviously with respect to Tables 5A and 5B.

TABLE 16A Residues that Define the Virginiamycin M Binding Pocket (5.8 Åshell) Corre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA A2100^(v) A2059 A3630A3874 A1052 G2102^(h,v) G2061 G3632 G3876 G1054 A2103^(h,v) A2062 A3633A3877 A1055 C2104^(h,v) C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879C1057 A2474 A2439 A4106 A4345 A1256 G2482^(v) G2447 G4114 G4353 G1264A2486^(v) A2451 A4118 A4357 A1268 C2487^(v) C2452 C4119 C4358 C1269U2535^(v) U2500 U4167 U4406 U1317 A2538^(h,v) A2503 A4170 A4409 A1320U2539^(v) U2504 U4171 U4410 U1321 G2540^(v) G2505 G4172 G4411 G1322U2541^(v) U2506 U4173 U4412 U1323 U2620^(v) U2585 U4252 U4491 U1402 Theresidues that define the binding pocket (5.8 Å shell) were determined asthose residues in the 50S ribosomal subunit that are located within 5.8angstroms of the atoms of virginiamycin M using the program MIDAS^(a).Conserved residues in 23S rRNA were determined by comparison ofsequences # from the structure of H. marismortui 23S rRNA^(b) with thecorresponding sequences of aligned genomic DNA encoding the homologous23S rRNA (E. coli ^(c), Rattus norvegicus ^(d), Human^(e) or Humanmitochondria^(f)). Sequence alignments were determined with the program# MegAlign (DNASTAR, Madison, Wisconsin, USA) using default parameters.^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988)“The MIDAS Display System” J. Mol. Graphics, 6(1):13-27, 36-37.^(b)GenBank accession AF034619 ^(c)GenBank accession J01695 ^(d)GenBankaccession 2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession13683 ^(h)Indicates residue that is within hydrogen-bonding distance(3.5 Å) of the ligand; for example, see Jorgensen, W L, Nguycn, T B. J.Comput. Chem. (1993) 14:195-205. ^(v)Residues in van der Waals contactwith the antibiotic are defined as those residues within rmin of anyatom in the antibiotic, where rmin = 21/6s and is the minimum in theLennard-Jones pair potential (see Computer Simulation of Liquids; Alien,M. P. and Tildesley, D. J.; Oxford # University Press: New York, 1992,11.). The Lennard-Jones ss are taken from the OPLS-AA force field (see,Jorgensen, W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc.,1996, 118, 11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; YaleUniversity: New Haven, CT, 2002).

TABLE 16B Residues that Define the Virginiamycin M Binding Pocket (5.8Å-12.6 Å shell) Corre- Corre- sponding sponding Corre- Corre- Residue inResidue sponding sponding Human H. marismortui in E. Residue in Residuein Mitochon- Residue coli Rattus Human dria 23S rRNA A631 A574 G1282G1363 NP* A632 A575 C1283 C1364 NP C633 U576 G1284 G1365 NP U1838 U1782U3369 U3613 C832 G2073 G2032 C3603 C3847 G1025 A2095 A2054 G3625 G3869A1047 A2096 C2055 A3626 A3870 C1048 G2097 G2056 A3627 A3871 G1049 C2098G2057 A3628 A3872 A1050 G2099 A2058 G3629 G3873 G1051 A2101 A2060 A3631A3875 A1053 C2106 C2065 C3636 C3880 C1058 U2107 C2066 U3637 U3881 U1059G2284 G2251 G3917 G4156 G1145 U2473 U2438 U4105 U4344 U1255 C2475 C2440C4107 C4346 C1257 C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109 A4348C1259 U2478 C2443 C4110 C4349 U1260 A2479 G2444 A4111 A4350 A1261 G2480G2445 G4112 G4351 G1262 G2481 G2446 G4113 G4352 G1263 U2484 U2449 U4116U4355 U1266 A2485 A2450 A4117 A4356 A1267 A2488 A2453 U4120 U4359 A1270G2489 G2454 G4121 G4360 G1271 A2532 A2497 C4164 C4403 A1314 C2534 C2499U4166 U4405 C1316 C2536 C2501 C4168 C4407 C1318 G2537 G2502 G4169 G4408G1319 C2542 C2507 C4174 C4413 U1324 U2607 A2572 A4239 A4478 A1389 C2608C2573 C4240 C4479 C1390 U2610 C2575 U4242 U4481 U1392 G2611 G2576 G4243G4482 G1393 A2612 A2577 A4244 A4483 A1394 G2618 G2583 G4250 G4489 G1400U2619 U2584 U4251 U4490 U1401 U2621 U2586 U4253 U4492 C1403 A2622 A2587A4254 A4493 A1404 C2636 C2601 C4268 C4507 C1418 A2637 A2602 A4269 A4508A1419 G2643 G2608 G4275 G4514 G1425 C2644 U2609 U4276 U4515 U1426 U2645C2610 U4277 U4516 U1427 G2646 C2611 U4278 U4517 U1428 L3 W242 Q150 W257W257 G246 The residues that define the binding pocket (5.8 Å-12.6 Åshell) were determined as those residues in the 50S ribosomal subunitthat are located within 5.8-12.6 angstroms of the atoms of virginiamycinM using the program MIDAS^(a). Conserved residues in 23S rRNA were #determined by comparison of sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). In # cases ofribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Human^(k)mitochondria^(l). Sequence # alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) with default parameters. *NPmeans that no homologous residue has been identified. ^(a)T. E. Ferrin,C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDAS DisplaySystem” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBank accessionAF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession 2624399^(e)GenBank accession M11167 ^(f)GenBank accession 13683 ^(g)GenBankaccession AAA86859 for protein L3. ^(i)GenBank accession CAA26460 forprotein L3. ^(j)GenBank accession P21531 for protein L3. ^(k)GenBankaccession NP_000958 for protein L3. ^(l)GenBank accession P09001 forprotein L3.

Table 17A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of a spiramycin bindingpocket (5.8 Å shell). In addition, Table 17A identifies thecorresponding residues that define at least a portion of the spiramycinbinding pocket in E. coli, Rattus, human, and human mitochondria largesubunit. Further, Table 17B identifies the residues in the H.marismortui 50S ribosomal subunit that together define a broader portionof a spiramycin binding pocket (5.8 Å-12.6 Å shell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 17A Residues that Define the Spiramycin Binding Pocket (5.8 Åshell) Corre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C839^(v) U746 G1495G1574 NP* C2098 G2057 A3628 A3872 A1050 G2099^(h,v) A2058 G3629 G3873G1051 A2100^(v) A2059 A3630 A3874 A1052 G2102^(v) G2061 G3632 G3876G1054 A2103^(h,v) A2062 A3633 A3877 A1055 A2538^(v) A2503 A4170 A4409A1320 U2539^(v) U2504 U4171 U4410 U1321 G2540^(h,v) G2505 G4172 G4411G1322 U2541 U2506 U4173 U4412 U1323 U2620^(m,v) U2585 U4252 U4491 U1402C2644 U2609 U4276 U4515 U1426 G2646^(v) C2611 U4278 U4517 U1428 L4S63^(v) S55 T69 T69 Q74 G64^(v) G56 G70 G70 E75 R65^(v) K57 R71 R71 R76L22 M130^(m, v) K90 H133 H133 V187 The residues that define the bindingpocket (5.8 Å shell) were determined as those residues in the 50Sribosomal subunit that are located within 5.8 angstroms of the atoms ofspiramycin using the program MIDAS^(a). Conserved residues weredetermined by comparison between the proposed # secondary structures ofH. marismortui ^(b), E. coli ^(c), and Rattus norvegicus ^(d),Human^(e), and Human Mitochondria^(e). Sequence alignments weredetermined with the program MegAlign (DNASTAR, Madison, Wisconsin, USA)using defaul parameters. *NP means that # no homologous residue has beenidentified. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R.Langridge (1988) “The MIDAS Display System” J. Mol. Graphics,6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBank accessionJ01695 ^(d)GenBank accession 2624399 ^(e)GenBank accession M11167^(f)GenBank accession 13683 ^(g)GenBank accession P12735 for protein L4;GenBank accession R5HS24 for protein L22. ^(h)Indicates residue that iswithin hydrogen-bonding distance (3.5 A) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(i)GenBank accession CAA26461 for protein L4; GenBank accessionCAA26465 for protein L22. ^(j)GenBank accession JC4277 for protein L4;GenBank accession P24049 for protein L22 ^(k)GenBank accessions P36578,NP_000959, S39803, and T09551 for protein L4; GenBank accessionXP_051279 for protein L22. ^(l)GenBank accession XP_049502 for proteinL4; GenBank accession XP_051279 for protein L22. ^(m)Residue U2620 isactually at 5.83 Å; M130 is actually at 5.87 Å. ^(v)Residues in van derWaals contact with the antibiotic are defined as those residues withinrmin of any atom in the antibiotic, where rmin = 21/6s and is theminimum in the Lennard-Jones pair potential (see Computer Simulation ofLiquids; Alien, M. P. and Tildesley, D. J.; Oxford # University Press:New York, 1992, 11.). The Lennard-Jones ss are taken from the OPLS-AAforce field (see, Jorgensen, W. L.; Maxwell, D. S. and Tirado-Rives, J.J. Am. Chem. Soc., 1996, 118, 11225-11236 and Jorgensen, W. L. BOSS,Version 4.3; Yale University: New Haven, CT, 2002).

TABLE 17B Residues that Define the Spiramycin Binding Pocket (5.8 Å-12.6Å shell) Corre- Corre- sponding sponding Corre- Corre- Residue inResidue sponding sponding Human H. marismortui in E. Residue in Residuein Mitochon- Residue coli Rattus Human dria 23S rRNA A766 A675 A1424A1502 NP* A767 A676 A1425 A1503 NP U835 U744 U1491 U1570 NP G836 G745G1492 G1571 NP U837 NP A1493 A1572 NP C838 NP C1494 C1573 NP U840 U747U1496 U1575 NP A841 G748 G1497 G1576 NP A843 A750 A1499 A1578 NP A844A751 A1500 A1579 NP U845 A752 A1501 A1580 NP A846 A753 U1502 U1581 NPA882 A789 A1538 A1617 NP U883 U790 U1539 U1618 NP C884 C791 C1540 C1619NP U1359 U1255 U2192 U2327 U351 G1837 U1781 A3368 A3612 C831 U1838 U1782U3369 U3612 C832 C2056 A2015 A3587 A3831 A1008 U2057 U2016 C3588 C3832U1009 G2073 G2032 C3603 C3847 G1025 A2095 A2054 G3625 G3869 A1047 G2097G2056 A3627 A3871 G1049 A2101 A2060 A3631 A3875 A1053 C2104 C2063 C3634C3878 C1056 C2105 C2064 C3625 C3879 C1057 A2474 A2439 A4106 A4345 A1256C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109 A4348 C1259 U2478 C2443C4110 C4349 U1260 A2479 G2444 A4111 A4350 A1261 G2481 G2446 G4113 G4352G1263 G2482 G2447 G4114 G4353 G1264 A2485 A2450 A4117 A4356 A1267 A2486A2451 A4118 A4357 A1268 C2487 C2452 C4119 C4358 C1269 A2488 A2453 U4120U4359 A1270 U2535 U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407 C1318G2537 G2502 G4169 G4408 G1319 C2542 C2507 C4174 C4413 U1324 U2607 A2572A4239 A4478 A1389 C2608 C2573 C4240 C4479 C1390 U2610 C2575 U4242 U4481U1392 G2611 G2576 G4243 G4482 G1393 A2612 A2577 A4244 A4483 A1394 C2614C2579 C4246 C4485 C1396 U2615 U2580 U4247 U4486 U1397 G2616 G2581 G4248G4487 G1398 G2618 G2583 G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401U2621 U2586 U4253 U4492 C1403 G2643 G2608 G4275 G4514 G1425 U2645 C2610U4277 U4516 U1427 C2647 C2612 U4279 U4518 C1429 U2648 U2613 A4280 A4519U1430 Protein L3 W242 Q150 W257 W257 G246 Protein L4 S60 V52 S66 S66 G71F61 T53 W67 W67 F72 G62 G54 G68 G68 E73 G66 NP A72 A72 G78 Q67 NP V73V73 L79 A68 NP A74 A74 A80 H69 K58 R75 R75 D81 Protein L22 A129 A89 A132A132 A186 G131 G91 G134 G134 A188 R132 R92 R135 R135 H189 The residuesthat define the binding pocket (5.8 Å-12.6 Å shell) were determined asthose residues in the 50S ribosomal subunit that are located within5.8-12.6 angstroms of the atoms of spiramycin using the programMIDAS^(a). Conserved residues in 23S rRNA were # determined bycomparison of sequences from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In # cases of ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human mitochondria^(l). Sequence #alignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. *NP means that no homologousresidue has been identified. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1): 13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession AAA86859 forprotein L3; GenBank accession P12735 for protein L4; GenBank accessionR5HS24 for protein L22. ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(i)GenBank accession CAA26460 for protein L3; GenBank accessionCAA26461 for protein L4; GenBank accession CAA26465 for protein L22.^(j)GenBank accession P21531 for protein L3; GenBank accession JC4277for protein L4; GenBank accession P24049 for protein L22. ^(k)GenBankaccession NP_000958 for protein L3; GenBank accessions P36578,NP_000959, S39803, and T09551 for protein LA; GenBank accessionXP_051279 for protein L22. ^(l)GenBank accession P09001 for protein L3;GenBank accession XP_049502 for protein L4; GenBank accession XP_051279for protein L22. ^(v)Residues in van der Waals contact with theantibiotic are defined as those residues within rmin of any atom in theantibiotic, where rmin = 21/6s and is the minimum in the Lennard-Jonespair potential (see Computer Simulation of Liquids; Alien, M. P. andTildesley, D. J.; Oxford # University Press: New York, 1992, 11.). TheLennard-Jones ss are taken from the OPLS-AA force field (see, Jorgensen,W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118,11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; Yale University: NewHaven, CT, 2002).

Table 18A identifies the residues in the H. marismortui 50S ribosomalsubunit that together define at least a portion of an erythromycinbinding pocket (5.8 Å shell). In addition, Table 18A identifies thecorresponding residues that define at least a portion of theerythromycin binding pocket in E. coli, Rattus, human, and humanmitochondria large subunit. Further, Table 18B identifies the residuesin the H. marismortui 50S ribosomal subunit that together define abroader portion of a erythromycin binding pocket (5.8 Å-12.6 Å shell).The non-conserved residues were identified as described previously withrespect to Tables 5A and 5B.

TABLE 18A Residues that Define the Erythromycin Binding Pocket (5.8 Åshell) Corre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria C839^(v) 0746 G1495 G1574 NP*A841^(v) G748 G1497 G1576 NP C2098^(h,v) G2057 A3628 A3872 A1050G2099^(h,v) A2058 G3629 G3873 G1051 A2100^(v) A2059 A3630 A3874 A1052A2103^(v) A2062 A3633 A3877 A1055 A2538^(v) A2503 A4170 A4409 A1320G2540^(v) G2505 G4172 G4411 G1322 C2644^(v) U2609 U4276 U4515 U1426U2645^(v) C2610 U4277 U4516 U1427 G2646^(v) C2611 U4278 U4517 U1428 L4G64^(v) G56 G70 G70 E75 L22 M130^(m,v) K90 H133 H133 V187 The residuesthat define the binding pocket (5.8 Å shell) were determined as thoseresidues in the 50S ribosomal subunit that are located within 5.8angstroms of the atoms of erythromycin using the program MIDAS^(a).Conserved residues in 23S rRNA were determined by comparison of #sequences from the structure of H. marismortui 23S rRNA^(b) with thecorresponding sequences of aligned genomic DNA encoding the homologous23S rRNA (E. coli ^(c), Rattus norvegicus ^(d), Human^(e) or Humanmitochondria^(f)). In cases of ribosomal proteins, comparisons # werealso made between the H. marismortui structural protein sequence^(g) andthe corresponding sequences of aligned protein sequences encodinghomologous proteins in E. coli ^(i), Rattus norvegicus ^(j), Human^(k)or Human mitochondria^(l). Sequence alignments were # determined withthe program MegAlign (DNASTAR, Madison, Wisconsin, USA) using defaultparameters. *NP means that no homologous residue has been identified.^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988)“The MIDAS Display System” J. Mol. Graphics, 6(1): 13-27, 36-37.^(b)GenBank accession AF034619 ^(c)GenBank accession J01695 ^(d)GenBankaccession 2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession13683 ^(g)GenBank accession P12735 for protein L4; GenBank accessionR5HS24 for protein L22. ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(i)GenBank accession CAA26461 for protein L4; GenBank accessionCAA26465 for protein L22. ^(j)GenBank accession JC4277 for protein L4;GenBank accession P24049 for protein L22 ^(k)GenBank accessions P36578,NP_000959, S39803, and T09551 for protein L4; GenBank accessionXP_051279 for protein L22. ^(l)GenBank accession XP_049502 for proteinL4; GenBank accession XP_051279 for protein L22. ^(m)Residue C2098 isactually at 5.99 Å; M130 is at 5.90 Å. ^(v)Residues in van der Waalscontact with the antibiotic are defined as those residues within rmin ofany atom in the antibiotic, where rmin = 21/6s and is the minimum in theLennard-Jones pair potential (see Computer Simulation of Liquids; Alien,M. P. and Tildesley, D. J.; Oxford # University Press: New York, 1992,11.). The Lennard-Jones ss are taken from the OPLS-AA force field (see,Jorgensen, W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc.,1996, 118, 11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; YaleUniversity: New Haven, CT. 2002).

TABLE 18B Residues that Further Define the Erythromycin Binding PocketCorre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C633 U576 G1284 G1365NP* U835 U744 U1491 U1570 NP G836 G745 G1492 G1571 NP U837 NP A1493A1572 NP C838 NP C1494 C1573 NP U840 U747 U1496 U1575 NP A843 A750 A1499A1578 NP A844 A751 A1500 A1579 NP U845 A752 A1501 A1580 NP A846 A753U1502 U1581 NP C847 U754 C1503 C1582 NP U1359 U1255 U2191 U2327 U351G1837 U1781 A3368 A3612 C831 U1838 U1782 U3369 U3613 C832 C2056 A2015A3587 A3831 A1008 U2057 U2016 C3588 C3832 U1009 A2096 C2055 A3626 A3870C1048 G2097 G2056 A3627 A3871 G1049 A2101 A2060 A3631 A3875 A1053 G2102G2061 G3632 G3876 G1054 C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635C3879 C1057 A2474 A2439 A4106 A4345 A1256 C2476 U2441 C4108 C4347 C1258C2477 C2442 A4109 A4348 C1259 U2478 C2443 C4110 C4349 U1260 G2482 G2447G4114 G4353 G1264 A2486 A2451 A4118 A4357 A1268 C2487 C2452 C4119 C4358C1269 U2535 U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407 C1318 G2537G2502 G4169 G4408 G1319 U2539 U2504 U4171 U4410 U1321 U2541 U2506 U4173U4412 U1323 C2542 C2507 C4174 C4413 U1324 G2543 G2508 G4175 G4414 G1325G2611 G2576 G4243 G4482 G1393 A2612 A2577 A4244 A4483 A1394 G2613 G2578G4245 G4484 U1395 C2614 C2579 C4246 C4485 C1396 U2615 U2580 U4247 U4486U1397 G2616 G2581 G4248 G4487 G1398 G2617 G2582 G4249 G4488 A1399 G2618G2583 G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401 U2620 U2585 U4252U4491 U1402 U2621 U2586 U4253 U4492 C1403 A2622 A2587 A4254 A4493 A1404G2643 G2608 G4275 G4514 G1425 C2647 C2612 U4279 U4518 C1429 U2648 U2613A4280 A4519 U1430 Protein L3 W242 Q150 W257 W257 G246 Protein L4 G62 G54G68 G68 E73 S63 S55 T69 T69 Q74 R65 K57 R71 R71 R76 Q67 NP* A72 A72 G78Protein L22 A129 A89 A132 A132 A186 G131 G91 G134 G134 A188 R132 R92R135 R135 H189 The residues that define the binding pocket (5.8 Å-12.6 Åshell) were determined as those residues in the 50S ribosomal subunitthat are located within 5.8-12.6 angstroms of the atoms of erythromycinusing the program MIDAS⁸. Conserved residues in 23S rRNA were determined# by comparison of sequences from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Ratttis norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In cases of # ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human mitochondria^(l). Sequence #alignments were determined with the program MegAlign (DNASTAR, Madison,Wisconsin, USA) using default parameters. *NP means that no homologousresidue has been identified. ^(a)T. E. Ferrin, C. C. Huang, L. E.Jarvis, and R. Langridge (1988) “The MIDAS Display System” J. Mol.Graphics, 6(1): 13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession AAA86859 forprotein L3; GenBank accession P12735 for protein L4; GenBank accessionR5HS24 for protein L22. ^(h)Indicates residue that is withinhydrogen-bonding distance (3.5 Å) of the ligand; for example, seeJorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(i)GenBank accession CAA26460 for protein L3; GenBank accessionCAA26461 for protein L4; GenBank accession CAA26465 for protein L22.^(j)GenBank accession P21531 for protein L3; GenBank accession JC4277for protein L4; GenBank accession P24049 for protein L22. ^(k)GenBankaccession NP_000958 for protein L3; GenBank accessions P36578,NP_000959, S39803, and T09551 for protein L4; GenBank accessionXP_051279 for protein L22. ^(l)GenBank accession P09001 for protein L3;GenBank accession XP_049502 for protein L4; GenBank accession XP_051279for protein L22. ^(v)Residues in van der Waals contact with theantibiotic are defined as those residues within rmin of any atom in theantibiotic, where rmin = 21/6s and is the minimum in the Lennard-Jonespair potential (see Computer Simulation of Liquids; Alien, M. P. andTildesley, D. J.; Oxford # University Press: New York, 1992, 11.). TheLennard-Jones ss are taken from the OPLS-AA force field (see, Jorgensen,W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118,11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; Yale University: NewHaven. CT, 2002).

Table 19A identifies the residues in the H marismortui 50S ribosomalsubunit that together define at least a portion of an azithromycinbinding pocket (5.8 Å shell). In addition, Table 19A identifies thecorresponding residues that define at least a portion of theazithromycin binding pocket in E. coli, Rattus, human, and humanmitochondria large subunit. Table 19B identifies the residues in the H.marismortui 50S ribosomal subunit that together define a broader portionof a azithromycin binding pocket (5.8 Å-12.6 Åshell). The non-conservedresidues were identified as described previously with respect to Tables5A and 5B.

TABLE 19A Residues that Define the Azithromycin Binding Pocket (5.8 Åshell) Corre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C839^(v) U746 G1495G1574 NP* G2099^(h,v) A2058 G3629 G3873 G1051 A2100^(v) A2059 A3630A3874 A1052 A2103^(v) A2062 A3633 A3877 A1055 A2538^(v) A2503 A4170A4409 A1320 G2540^(v) G2505 G4172 G4411 G1322 U2541^(m,v) U2506 U4173U4412 U1323 C2644^(v) U2609 U4276 U4515 U1426 U2645^(v) C2610 U4277U4516 U1427 G2646^(v) C2611 U4278 U4517 U1428 Protein L22 M130^(m,v) K90H133 H133 V187 The residues that define the binding pocket (5.8 Å shell)were determined as those residues in the 50S ribosomal subunit that arelocated within 5.8 angstroms of the atoms of azithromycin using theprogram MIDAS^(a). Conserved residues in 23S rKNA were determined bycomparison of sequences # from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA (E. coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In cases of ribosomal proteins,comparisons were # also made between the H. marismortui structuralprotein sequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli ^(i), Rattusnorvegicus ^(j), Human^(k) or Human mitochondria^(l). Sequencealignments were # determined with the program MegAlign (DNASTAR,Madison, Wisconsin, USA) using default parameters. *NP means that nohomologous residue has been identified. ^(a)T. E. Ferrin, C. C. Huang,L. E. Jarvis, and R. Langridge (1988) ”The MIDAS Display System“ J. Mol.Graphics, 6(1): 13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBankaccession J01695 ^(d)GenBank accession 2624399 ^(e)GenBank accessionM11167 ^(f)GenBank accession 13683 ^(g)GenBank accession R5HS24 forprotein L22. ^(h)Indicates residue that is within hydrogen-bondingdistance (3.5 Å) of the ligand; for example, see Jorgensen, W L, Nguyen,T B. (1993) J. Comput. Chem. 14:195-205. ^(i)GenBank accession CAA26465for protein L22. ^(j)GenBank accession P24049 for protein L22^(k)GenBank accession XP_051279 for protein L22. ^(l)GenBank accessionXP_051279 for protein L22. ^(m)Residue U2541 is actually at 5.93 Å; M130is at 5.95 Å. ^(v)Residues in van der Waals contact with the antibioticare defined as those residues within rmin of any atom in the antibiotic,where rmin = 21/6s and is the minimum in the Lennard-Jones pairpotential (see Computer Simulation of Liquids; Alien, M. P. andTildesley, D. J.; Oxford # University Press: New York, 1992, 11.). TheLennard-Jones ss are taken from the OPLS-AA force field (see, Jorgensen,W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118,11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; Yale University: NewHaven, CT, 2002).

TABLE 19B Residues that Further Define the Azithromycin Binding PocketCorre- Corre- sponding sponding Corre- Corre- Residue in Residuesponding sponding Human H. marismortui in E. Residue in Residue inMitochon- Residue coli Rattus Human dria 23S rRNA C633 U576 G1284 G1365NP* U835 U744 U1491 U1570 NP G836 G745 G1492 G1571 NP U837 NP A1493A1572 NP C838 NP C1494 C1573 NP U840 U747 U1496 U1575 NP A841 G748 G1497G1576 NP A843 A750 A1499 A1578 NP A844 A751 A1500 A1579 NP U845 A752A1501 A1580 NP A846 A753 U1502 U1581 NP C847 U754 C1503 C1582 NP U1359U1255 U2191 U2327 U351 G1837 U1781 A3368 A3612 C831 U1838 U1782 U3369U3613 C832 C2056 A2015 A3587 A3831 A1008 U2057 U2016 C3588 C3832 U1009A2096 C2055 A3626 A3870 C1048 G2097 G2056 A3627 A3871 G1049 C2098 G2057A3628 A3872 A1050 A2101 A2060 A3631 A3875 A1053 G2102 G2061 G3632 G3876G1054 C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879 C1057 A2474A2439 A4106 A4345 A1256 C2476 U2441 C4108 C4347 C1258 C2477 C2442 A4109A4348 C1259 U2478 C2443 C4110 C4349 U1260 G2481 G2446 G4113 G4352 G1263G2482 G2447 G4114 G4353 G1264 A2486 A2451 A4118 A4357 A1268 C2487 C2452C4119 C4358 C1269 U2535 U2500 U4167 U4406 U1317 C2536 C2501 C4168 C4407C1318 G2537 G2502 G4169 G4408 G1319 U2539 U2504 U4171 U4410 U1321 C2542C2507 C4174 C4413 U1324 G2543 G2508 G4175 G4414 G1325 U2607 A2572 A4239A4478 A1389 G2611 G2576 G4243 G4482 G1393 A2612 A2577 A4244 A4483 A1394G2613 G2578 G4245 G4484 U1395 C2614 C2579 C4246 C4485 C1396 U2615 U2580U4247 U4486 U1397 G2616 G2581 G4248 G4487 G1398 G2617 G2582 G4249 G4488A1399 G2618 G2583 G4250 G4489 G1400 U2619 U2584 U4251 U4490 U1401 U2620U2585 U4252 U4491 U1402 U2621 U2586 U4253 U4492 C1403 A2622 A2587 A4254A4493 A1404 G2643 G2608 G4275 G4514 GI425 C2647 C2612 U4279 U4518 C1429U2648 U2613 A4280 A4519 U1430 Protein L3 W232 S139 NP NP T236 W242 Q150W257 W257 G246 Protein L4 G62 G54 G68 G68 E73 S63 S55 T69 T69 Q74 G64G56 G70 G70 E75 R65 K57 R71 R71 R76 G66 NP A72 A72 G78 Q67 NP V73 V73L79 Protein L22 A129 A89 A132 A132 A186 G131 G91 G134 G134 A188 R132 R92R135 R135 H189 The residues that define the binding pocket (5.8 Å-12.6 Åshell) were determined as those residues in the 50S ribosomal subunitthat are located within 5.8-12.6 angstroms of the atoms of azithromycinusing the program MIDAS^(a). Conserved residues in 23S rRNA were #determined by comparison of sequences from the structure of H.marismortui 23S rRNA^(b) with the corresponding sequences of alignedgenomic DNA encoding the homologous 23S rRNA (E. coli ^(c), Rattusnorvegicus ^(d), Human^(e) or Human mitochondria^(f)). In # cases ofribosomal proteins, comparisons were also made between the H.marismortui structural protein sequence^(g) and the correspondingsequences of aligned protein sequences encoding homologous proteins inE. coli ^(i), Rattus norvegicus ^(j), Human^(k) or Human #mitochondria^(l). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) with default parameters. *NPmeans that no homologous residue has been identified. ^(a)T. E. Ferrin,C. C. Huang, L. E. Jarvis, and R. Langridge (1988) “The MIDAS DisplaySystem” J. Mol. Graphics, 6(1):13-27, 36-37. ^(b)GenBank accessionAF034619 ^(c)GenBank accession J01695 ^(d)GenBank accession 2624399^(e)GenBank accession M11167 ^(f)GenBank accession 13683 ^(g)GenBankaccession AAA86859 for protein L3; GenBank accession P12735 for proteinL4; GenBank accession R5HS24 for protein L22. ^(h)Indicates residue thatis within hydrogen-bonding distance (3.5 Å) of the ligand; for example,see Jorgensen, W L, Nguyen, T B. (1993) J. Comput. Chem. 14:195-205.^(i)GenBank accession CAA26460 for protein L3; GenBank accessionCAA26461 for protein L4; GenBank accession CAA26465 for protein L22.^(j)GenBank accession P21531 for protein L3; GenBank accession JC4277for protein L4; GenBank accession P24049 for protein L22. ^(k)GenBankaccession NP_000958 for protein L3; GenBank accessions P36578,NP_000959, S39803, and T09551 for protein L4; GenBank accessionXP_051279 for protein L22. ^(l)GenBank accession P09001 for protein L3;GenBank accession XP_049502 for protein L4; GenBank accession XP_051279for protein L22. ^(v)Residues in van der Waals contact with theantibiotic are defined as those residues within rmin of any atom in theantibiotic, where rmin = 21/6 s and is the minimum in the Lennard-Jonespair potential (see Computer Simulation of Liquids; Alien, M. P. andTildesley, D. J.; Oxford # University Press: New York, 1992, 11.). TheLennard-Jones ss are taken from the OPLS-AA force field (see, Jorgensen,W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. Soc., 1996, 118,11225-11236 and Jorgensen, W. L. BOSS, Version 4.3; Yale University: NewHaven, CT, 2002).

Table 20A identifies the residues in the H. Marismortui 50S ribosomalsubunit that together define at least a portion of a linezolid bindingpocket (5.8 Å shell). In addition, Table 20A identifies thecorresponding residues that define at least a portion of the linezolidbinding pocket in E coli, Rattus, human, and human mitochondria largesubunit. Table 20B identifies the residues in the H. Marismortui 50Sribosomal subunit that together define a broader portion of a linezolidbinding pocket (5.8 Å-12.6 Å shell). The non-conserved residues wereidentified as described previously with respect to Tables 5A and 5B.

TABLE 20A Residues that Define the Linezolid Binding Pocket (5.8 Åshell) Corre- Corre- Corre- Corre- sponding H. marismortui spondingsponding sponding Residue in Residue Residue in Residue in Residue inHuman 23S rRNA E. coil Rattus Human Mitochondria A2l00^(v) A2059 A3630A3874 A1052 G2102^(v) G2061 G3632 G3876 G1054 A2103^(v) A2062 A3633A3877 A1055 G2482^(v) G2447 G4114 G4353 G1264 A2486^(v) A2451 A4118A4357 A1268 C2487^(h,v) C2452 C4119 C4358 C1269 A2488^(v) A2453 U4120U4359 A1270 U2535^(v) U2500 U4167 U4406 U1317 A2538^(v) A2503 A4170A4409 A1320 U2539^(h,v) U2504 U4171 U4410 U1321 G2540^(h,v) G2505 G4172G4411 G1322 U2541^(h,v) U2506 U4173 U4412 U1323 C2542^(v) C2507 C4174C4413 U1324 U2619^(v) U2584 U4251 U4490 U1401 U2620^(v) U2585 U4252U4491 U1402 The residues that define the binding pocket (5.8 Å shell)were determined as those residues in the 50S ribosomal subunit that arelocated within 5.8 angstroms of the atoms of linezolid using the programMIDAS^(a). Conserved residues in 23S rRNA were determined by comparisonof sequences from the structure of H. marismortui 23S rRNA^(b) with thecorresponding sequences of aligned genomic DNA encoding the homologous23S rRNA (E. coli ^(c), # Rattus norvegicus ^(d), Human^(c) or Humanmitochondria^(f)). Sequence alignments were determined with the programMegAlign (DNASTAR, Madison, Wisconsin, USA) with default parameters.^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R. Langridge (1988)“The MIDAS Display System” J. Mol. Graphics, 6(l): 13-27, 36-37.^(b)GenBank accession AF034619 ^(c)GenBank accession J01695 ^(d)GenBankaccession 2624399 ^(e)GenBank accession M11167 ^(f)GenBank accession13683 ^(h)Indicates residue that is within hydrogen-bonding distance(3.5 Å) of the ligand; for example, see Jorgensen, W L, Nguyen, T B.(1993) J. Comput. Chem. 14: 195-205. ^(v)Residues in van der Waalscontact with the antibiotic are defined as those residues within rmin ofany atom in the antibiotic, where rmin = 21/6s and is the minimum in theLennard-Jones pair potential (see Computer Simulation of Liquids; Allen,M. P. and Tildesley, D. J.; Oxford University Press: New York, 1992,11.). The Lennard-Jones ss are taken from the OPLS-AA force field (see,Jorgensen, W. L.; Maxwell, D. S. and Tirado-Rives, J. J. Am. Chem. #Soc., 1996, 118, 11225-11236 and Jorgensen, W. L. BOSS, Version 4.3;Yale University: New Haven, CT, 2002).

TABLE 20B Residues that Further Define the Linezolid Binding Pocket (5.8Å-12.6 Å shell) Corre- sponding Corre- Corre- Corre- Residue in H.sponding sponding sponding Human marismortui Residue E. Residue inResidue in Mito- Residue coli Rattus Human chondria 23s rRNA A631 A574G1282 G1363 NP* A632 A575 C1283 C1364 NP C633 U576 G1284 G1365 NP G2073G2032 C3603 C3847 G1025 G2094 G2053 G3624 G3868 G1046 A2095 A2054 G3625G3869 A1047 A2096 C2055 A3626 A3870 C1048 G2097 G2056 A3627 A3871 G1049C2098 G2057 A3628 A3872 A1050 G2099 A2058 G3629 G3873 G1051 A2101 A2060A3631 A3875 A1053 C2104 C2063 C3634 C3878 C1056 C2105 C2064 C3635 C3879C1057 A2474 A2439 A4106 A4345 A1256 G2480 G2445 G4112 G4351 G1262 G2481G2446 G4113 G4352 G1263 A2485 A2450 A4117 A4356 A1267 G2489 G2454 G4121G4360 G1271 U2528 U2493 U4160 U4399 U1310 G2529 G2494 G4161 G4400 A1311C2534 C2499 U4166 U4405 C1316 C2536 C2501 C4168 C4407 C1318 G2537 G2502G4169 G4408 G1319 G2543 G2508 G4175 G4414 G1325 G2588 G2553 G4220 G4459G1370 U2607 A2572 A4239 A4478 A1389 C2608 C2573 C4240 C4479 C1390 G2609G2574 G4241 G4480 G1391 U2610 C2575 U4242 U4481 U1392 G2611 G2576 G4243G4482 G1393 A2612 A2577 A4244 A4483 A1394 G2616 G258l G4248 G4487 G1398G2617 G2582 G4249 G4488 A1399 G2618 G2583 G4250 G4489 G1400 U2621 U2586U4253 U4492 C1403 A2637 A2602 A4269 A4508 A1419 U2645 C2610 U4277 U4516U1427 G2646 C2611 U4278 U4517 U1428 Protein L3 W242 Q150 W257 W257 G246The residues that define the binding pocket (5.8 Å-12.6 Å shell) weredetermined as those residues in the 50S ribosomal subunit that arelocated within 5.8-12.6 angstroms of the atoms of linezolid using theprogram MIDAS^(a). Conserved residues in 23S rRNA were determined bycomparison of sequences from the structure of H. marismortui 23SrRNA^(b) with the corresponding sequences of aligned genomic DNAencoding the homologous 23S rRNA # (E.coli ^(c), Rattus norvegicus ^(d),Human^(e) or Human mitochondria^(f)). In cases of ribosomal proteins,comparisons were also made between the H. marismortui structural proteinsequence^(g) and the corresponding sequences of aligned proteinsequences encoding homologous proteins in E. coli, Rattus norvegicus^(j), Human^(k) or Human mitochondria^(l). Sequence alignments weredetermined with the program MegAlign (DNASTAR, Madison, # Wisconsin,USA)with default parameters. *NP means that no homologous residue hasbeen identified. ^(a)T. E. Ferrin, C. C. Huang, L. E. Jarvis, and R.Langridge (1988) “The MIDAS Display System” J. Mol. Graphics,6(1):13-27, 36-37. ^(b)GenBank accession AF034619 ^(c)GenBank accession101695 ^(d)GenBank accession 2624399 ^(e)GenBank accession M11167^(f)GenBank accession 13683 ^(g)GenBank accession AAA 86859 for proteinL3. ^(i)GenBank accession CAA26460 for protein L3. ^(j)GenBank accessionP21531 for protein L3. ^(k)GenBank accession NP_000958 for protein L3.^(l)GenBank accession P09001 for protein L3.

The skilled artisan, when in possession of the foregoing or otherexemplary target sites, may use the process of rational drug design toidentify molecules that potentially bind to one or more of the targetsites and/or inhibit ribosomal activity. Furthermore, by taking intoaccount which of the residues that define the target site are conservedbetween pathogens but not conserved between host species, the skilledartisan can design new species-specific protein synthesis inhibitors. Itis apparent that the skilled artisan can take advantage of the regionsthat are not conserved between E. coli and rat or human to providetarget regions for rational drug design. By way of example, FIG. 33shows certain regions of the polypeptide exit tunnel that are conservedbetween E. coli and rat or human (denoted in red) and regions of thepolypeptide exit tunnel that are not conserved between E. coli and rator human (denoted in blue). FIGS. 33(A) and 33(B) provide enlarged viewsof a large ribosomal subunit when cut in half along the polypeptide exittunnel. FIG. 33(C) is provided to orient the reader to the view in FIG.33(A) relative to the large ribosomal subunit. FIG. 33(D) is provided toorient the reader to the view in FIG. 33B relative to the largeribosomal subunit. In addition, the skilled artisan when in possessionof mutations that prevent or reduce antibiotic activity (i.e., arerelated to antibiotic resistance) can use this information to model therelevant antibiotic binding product which can then be used as a basisfor rational drug design to identify small molecules that overcome drugresistance. It is contemplated that a variety of computer modelingprocedures, for example, homology modeling protocols, can be used toprovide a model of a drug resistance target site by implementing sitedirected mutagenesis of nucleotides and/or amino acids and then usingthe appropriate energy minimization and refinement protocols.

c. Identification of Candidate Molecules.

It is contemplated that candidate molecules that inhibit proteinbiosynthesis can be designed entirely de novo or may be based upon apre-existing protein biosynthesis inhibitor. Either of these approachescan be facilitated by computationally screening databases and librariesof small molecules for chemical entities, agents, ligands, or compoundsthat can bind in whole, or in part, to ribosomes and ribosomal subunits,more preferably to large ribosomal subunits, and even more preferably to50S ribosomal subunits. In this screening, the quality of fit of suchentities or compounds to the binding site or sites may be judged eitherby shape complementarity or by estimated interaction energy (Meng et al.(1992) J. Comp. Chem. 13: 505-524).

The design of molecules that bind to or inhibit the functional activityof ribosomes or ribosomal subunits according to this invention generallyinvolves consideration of two factors. First, the molecule must becapable of physically and structurally associating with the largeribosomal subunit. Non-covalent molecular interactions important in theassociation of ribosomes and ribosomal subunits with the molecule,include hydrogen bonding, van der Waals and hydrophobic interactions.Second, the molecule must be able to assume a conformation that allowsit to associate with the ribosomes or ribosomal subunits, morepreferably with the large ribosomal subunits, and even more preferablywith the 50S ribosomal subunit. Although certain portions of themolecule may not directly participate in this association with aribosome or ribosomal subunits those portions may still influence theoverall conformation of the molecule. This, in turn, may have asignificant impact on binding affinities, therapeutic efficacy,drug-like qualities, and potency. Such conformational requirementsinclude the overall three-dimensional structure and orientation of thechemical entity or molecule in relation to all or a portion of theactive site or other region of the ribosomes or ribosomal subunits, orthe spacing between functional groups of a molecule comprising severalchemical entities that directly interact with the ribosomes or ribosomalsubunits, more preferably with the large ribosomal subunits, and evenmore preferably with the 50S ribosomal subunit.

The potential, predicted, inhibitory or binding effect of a molecule onribosomes and ribosomal subunits may be analyzed prior to its actualsynthesis and testing by the use of computer modeling techniques. If thetheoretical structure of the given molecule suggests insufficientinteraction and association between it and ribosomes or ribosomalsubunits, synthesis and testing of the molecule is obviated. However, ifcomputer modeling indicates a strong interaction, the molecule may thenbe synthesized and tested for its ability to interact with the ribosomesor ribosomal subunits and inhibit protein synthesis. In this manner,synthesis of inoperative molecules may be avoided. In some cases,inactive molecules are synthesized predicted on modeling and then testedto develop a SAR (structure-activity relationship) for moleculesinteracting with a specific region of the ribosome or ribosomal subunit,more preferably of the large ribosomal subunit, and even more preferablyof the 50S ribosomal subunit. As used herein, the term “SAR”, shallcollectively refer to the structure-activity/structure propertyrelationships pertaining to the relationship(s) between a compound'sactivity/properties and its chemical structure.

d. De Novo Design.

One skilled in the art may use one of several methods to identifychemical moieties or entities, compounds, or other agents for theirability to associate with a preselected target site within a ribosomesor ribosomal subunit. This process may begin by visual inspection orcomputer assisted modeling of, for example, the target site on thecomputer screen based on the atomic co-ordinates of the 50S ribosomalsubunit and/or its complexes with other analogues and antibiotics,deposited in the RCSB Protein Data Bank with accession numbers PDB ID:1FFK, 1JJ2, 1FFZ, 1FG0, 1K73, 1KC8, 1K8A, 1KD1, or 1K9M, and/orcontained on Disk N one embodiment, compound design uses computermodeling programs which calculate how different molecules interact withthe various sites of the ribosome, ribosomal subunit, or a fragmentthereof. Selected chemical moieties or entities, compounds, or agentsmay then be positioned in a variety of orientations, or docked, withinat least a portion of the target site of a ribosome or ribosomalsubunit, more preferably of a large ribosomal subunit, and even morepreferably of a 50S ribosomal subunit. Databases of chemical structuresare available from, for example, Cambridge Crystallographic Data Center(Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).Docking may be accomplished using software such as Cerius, Quanta orSybyl, followed by energy minimization and molecular dynamics withstandard molecular mechanics forcefields, such as OPLS-AA, CHARMM orAMBER.

Specialized computer programs may also assist in the process ofselecting chemical entities. These include, but are not limited to:

(1) GRID (Goodford, P. J., “A Computational Procedure for DeterminingEnergetically Favorable Binding Sites on Biologically ImportantMacromolecules” (1985) J. Med. Chem. 28, 849-857). Software such asGRID, a program that determines probable interaction sites betweenprobes with various functional group characteristics and themacromolecular surface, can be used to analyze the surface sites todetermine structures of similar inhibiting proteins or molecules. TheGRID calculations, with suitable inhibiting groups on molecules (e.g.,protonated primary amines) as the probe, are used to identify potentialhotspots around accessible positions at suitable energy contour levels.GRID is available from Oxford University, Oxford, UK.

(2) MCSS (Miranker, A. and M. Karplus (1991) “Functionality Maps ofBinding Sites: A Multiple Copy Simultaneous Search Method.” Proteins:Structure, Function and Genetics 11: 29-34). MCSS is available fromMolecular Simulations, Burlington, Mass.

(3) AUTODOCK (Goodsell, D. S. and A. J. Olsen (1990) “Automated Dockingof Substrates to Proteins by Simulated Annealing” Proteins: Structure,Function, and Genetics 8: 195-202). AUTODOCK is available from ScrippsResearch Institute, La Jolla, Calif.

(4) DOCK (Kuntz, I. D. et al. (1982) “A Geometric Approach toMacromolecule-Ligand Interactions” J. Mol. Biol. 161: 269-288). Theprogram DOCK may be used to analyze an active site or ligand bindingsite and suggest ligands with complementary steric properties. DOCK isavailable from University of California, San Francisco, Calif.

(5) ALADDIN (Van Drie et al. (1989) “ALADDIN: An Integrated Tool ofComputer Assisted Molecular Design and Pharmacophore Recognition FromGeometric, Steric and Substructure Searching of Three-DimensionalStructures” J. Comp-Aided Mol. Des. 3: 225).

(6) CLIX (Davie and Lawrence (1992) “CLIX: A Search Algorithm forFunding Novel Ligands Capable of Binding Proteins of KnownThree-Dimensional Structure” Proteins 12: 31-41).

(7) GROUPBUILD (Rotstein and Murcko (1993) “GroupBuild: A Fragment-BasedMethod for De Novo Drug Design”J. Med. Chem 36: 1700).

(8) GROW (Moon and Howe (1991) “Computer Design of Bioactive Molecules:A Method for Receptor-Based De Novo Ligand Design” Proteins 11: 314).

Once suitable chemical moieties or entities, compounds, or agents havebeen selected, they can be assembled into a single molecule. Assemblymay proceed by visual inspection and/or computer modeling andcomputational analysis of the spatial relationship of the chemicalmoieties or entities, compounds or agents with respect to one another inthree-dimensional space. This could then be followed by model buildingusing software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting theindividual chemical entities, compounds, or agents include but are notlimited to:

(1) CAVEAT (Bartlett, P. A. et al. (1989) “CAVEAT: A Program toFacilitate the Structure-Derived Design of Biologically ActiveMolecules”. In molecular Recognition in Chemical and BiologicalProblems”, Special Pub., Royal Chem. Soc. 78: 82-196) and (Bacon et al.(1992) J. Mol. Biol. 225: 849-858). CAVEAT uses databases of cycliccompounds which can act as “spacers” to connect any number of chemicalfragments already positioned in the active site. This allows one skilledin the art to quickly generate hundreds of possible ways to connect thefragments already known or suspected to be necessary for tight binding.CAVEAT is available from the University of California, Berkeley, Calif.

(2) 3D Database systems such as MACCS-3D (MDL Information Systems, SanLeandro, (Calif.). This area is reviewed in Martin, Y. C., (1992) “3DDatabase Searching in Drug Design”, J. Med. Chem. 35: 2145-2154.

(3) HOOK (available from Molecular Simulations, Burlington, Mass.).

Instead of proceeding to build a molecule of interest in a step-wisefashion one chemical entity at a time as described above, the moleculeof interest may be designed as a whole using either an empty active siteor optionally including some portion or portions of a known inhibitor orinhibitors. Software that implements these methods include:

(1) LUDI (Bohm, H.-J. (1992) “The Computer Program LUDI: A New Methodfor the De Novo Design of Enzyme Inhibitors” J. Comp. Aid. Molec. Design6: 61-78). The program LUDI can determine a list of interaction sitesinto which to place both hydrogen bonding and hydrophobic fragments.LUDI then uses a library of approximately 600 linkers to connect up tofour different interaction sites into fragments. Then smaller “bridging”groups such as —CH₂— and —COO— are used to connect these fragments. Forexample, for the enzyme DHFR, the placements of key functional groups inthe well-known inhibitor methotrexate were reproduced by LUDI. See also,Rotstein and Murcko, (1992) J. Med. Chem. 36:1700-1710. LUDI isavailable from Biosym Technologies, San Diego, Calif.

(2) LEGEND (Nishibata, Y. and A. Itai (1991) Tetrahedron 47, 8985).LEGEND is available from Molecular Simulations, Burlington, Mass.

(3) LeapFrog (available from Tripos Associates, St. Louis, Mo.).

(4) Aladdin (available from Daylight Chemical Information Systems,Irvine, Calif.)

Other molecular modeling techniques may also be employed in accordancewith this invention. See, e.g., Cohen, N. C. et al. (1990) “MolecularModeling Software and Methods for Medicinal Chemistry, J. Med. Chem. 33:883-894. See also, Navia, M. A. and M. A. Murcko (1992) “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology 2: 202-210; and Jorgensen (1998) “BOSS—Biochemical and OrganicSimulation System” in the Encyclopedia of Computational Chemistry (P. V.R. Schleyer, ed.) Wiley & Sonstra., Athens, U.S.A. 5: 3281-3285).

It is contemplated that during modeling, it may be possible to introduceinto the molecule of interest, chemical moieties that may be beneficialfor a molecule that is to be administered as a pharmaceutical. Forexample, it may be possible to introduce into or omit from the moleculeof interest, chemical moieties that may not directly affect binding ofthe molecule to the target area but which contribute, for example, tothe overall solubility of the molecule in a pharmaceutically acceptablecarrier, the bioavailability of the molecule and/or the toxicity of themolecule. Considerations and methods for optimizing the pharmacology ofthe molecules of interest can be found, for example, in “Goodman andGilman's The Pharmacological Basis of Therapeutics” Eighth Edition(Goodman, Gilman, Rail, Nies, & Taylor (eds.)). Pergaman Press (1985);Jorgensen & Duffy (2000) Bioorg. Med. Chem. Lett. 10: 1155-1158.

Furthermore, the computer program “Qik Prop” can be used to providerapid predictions for physically significant descriptions andpharmaceutically-relevant properties of an organic molecule of interest.A ‘Rule of Five’ probability scheme can be used to estimate oralabsorption of the newly synthesized compounds (Lipinski et al. (1997)Adv. Drug Deliv. Rev. 23:3).

Programs suitable for pharmacophore selection and design include:

(1) DISCO (Abbot Laboratories, Abbot Park, Ill.).

(2) Catalyst (Bio-CAD Corp., Mountain View, Calif.).

(3) Chem DBS-3D (Chemical Design Ltd., Oxford, U.K.).

Furthermore, the skilled artisan may use the information available onhow to design suitable therapeutically active and pharmaceuticallyuseful compounds, and use this information in the design of new proteinsynthesis inhibitors of the invention. See, for example, Lipinski et al.(1997) Ad Drug Deliv. Reviews 23: 3-25; Van de Waterbeemd et al. (1996)Quantitative Structure-Activity Relationships 15: 480-490; and Crucianiet al. (2000), Theochem-J. Mol. Struct. 503: 17-30.

The entry of the co-ordinates of the ribosome's or ribosomal subunit'sproteins and RNAs into the computer programs discussed above results inthe calculation of most probable structure of the macromolecule,including overall atomic co-ordinates of a ribosome, ribosomal subunitor a fragment thereof. These structures can be combined and refined byadditional calculations using such programs to determine the probable oractual three-dimensional structure of the ribosome, ribosomal subunit ora fragment thereof, including potential or actual active or bindingsites of ligands.

e. Modification of Existing Molecules.

Instead of designing molecules of interest entirely de novo it iscontemplated that pre-existing molecules or proteins thereof may be usedas a starting point for the design of a new candidate. It iscontemplated that many of the approaches useful for designing moleculesde novo may also be useful for modifying existing molecules.

It is contemplated that knowledge of the spatial relationship between aprotein biosynthesis inhibitor, for example, an antibiotic, and itsrespective binding site within a ribosome permits the design of modifiedinhibitors that may have better binding properties, for example, higherbinding affinity and/or specificity, relative to the molecule from whichit was derived. Alternatively, knowledge of inhibitor contact siteswithin a ribosome permits the synthesis of a new molecule that contain,for example, a portion of a first molecule that binds to the contactsite and another portion that contributes additional functionality.

It is contemplated that a variety of modified molecules (for example,modified antibiotics) may be designed using the atomic co-ordinatesprovided herein. For example, it is contemplated that by knowing thespatial relationship of one or more of antibiotics relative to the largeribosomal subunit it is possible to generate new antibiotic-basedmolecules. The atomic co-ordinates of each antibiotic relative to thelarge ribosomal subunit provides information on what portions of theribosome or ribosomal subunit and the antibiotic contact one another.Accordingly, from this information the skilled artisan may not onlyidentify contact locations within the ribosome that can be used for denovo drug design, as discussed above, but also may identify portions ofan antibiotic that can act as a ribosome binding domain.

Based on the information provided herein, the skilled artisan mayreadily identify and produce hybrid antibiotics that comprise a ribosomebinding domain of a first antibiotic and a ribosome binding domain of asecond, different antibiotic. The resulting hybrid antibioticspreferably bind to each of respective contact locations within theribosomal subunit simultaneously. The atomic co-ordinates providedherein permit the skilled artisan to identify candidate antibiotics thatmay be used as templates in the synthesis of a hybrid, and also providesteric information necessary to produce linking chemistries such thateach ribosome binding domain is properly orientated relative to itsrespective contact site. As a result, it is contemplated that theskilled artisan may produce a hybrid antibiotic that binds to a ribosomeor ribosomal subunit with a higher affinity and/or have higher proteinsynthesis inhibitory activity than either of the individual templateantibiotics used to generate the hybrid. Alternatively, the hybridantibiotic may overcome resistance phenotypes that may have developedagainst either of the template antibiotics. For example, the proximityof the site occupied by the disaccharide moiety of carbomycin to thesite filled by anisomycin suggests that a hybrid compound includingportions of both carbomycin and anisomycin may be an effective inhibitorof protein synthesis.

Furthermore, the atomic co-ordinates provided herein permit the skilledartisan to use the information pertaining to identify a ribosome bindingdomain and to design other types of protein synthesis inhibitors. Forexample, with an understanding of the ribosome contact region and thesurrounding environment, the skilled artisan can provide novelmolecules, a portion of which is based upon the antibiotic bindingregion (binding domain) and another portion of which (effector domain)can be designed as a novel space filling domain that sterically inhibitsor disrupts protein biosynthesis within the ribosome or secretionthrough the polypeptide exit tunnel. For example, the skilled artisanmay combine the ribosome binding region of the antibiotic, tylosin,which binds to one side of the polypeptide exit tunnel close to thepeptidyl transferase site, with, for example, a novel chemical moietynot present in antibiotics identified to date that is bulky enough toblock the polypeptide exit tunnel. However, it is contemplated that theskilled artisan may take advantage of one or more of the many of theantibiotic contact regions disclosed herein to design entirely newbinding and effector domains.

The resulting protein synthesis inhibitors have a molecular weight lessthan about 1,500, more preferably less than about 1,000, more preferablyless than 750 and, most preferably less than about 500. In addition, theprotein synthesis inhibitors have a minimal inhibitor concentration toinhibit 50% activity (IC₅₀) in a biological assay, for example, an invitro translation assay, for example, an E. coli translation assay.

Furthermore, the present invention permits the skilled artisan to designmolecules, for example, selective protein synthesis inhibitors that aretailored to be more potent with respect to ribosomes of a targetorganism, for example, a pathogen such a microbe, and less potent, i.e.,less toxic, to ribosomes of a non target organism, for example, hostorganism such as a human. Also, the invention permits the skilledartisan to use the atomic co-ordinates and structures of the largeribosomal subunit and its complexes with protein synthesis inhibitors todesign modifications to starting compounds, such as an antibiotic, thatwill bind more tightly to a target ribosome (e.g., the 50S ribosomalsubunit of bacteria) and less tightly to a non-targeted ribosome (e.g.,human 60S ribosomal subunit or a human mitochondrial ribosome).

The structure of a complex between the large ribosomal subunit and thestarting compound (e.g., tylosin or another protein synthesis inhibitor)can also be used to guide the modification of that compound to producenew compounds that have other desirable properties for the applicableindustrial and other uses (e.g., as pharmaceuticals, herbicides orinsecticides), such as chemical stability, solubility or membranepermeability.

A variety of antibiotics bind the large ribosomal subunit and disruptprotein synthesis and include members of antibiotic families whichinclude, but are not limited to: chloramphenicols, macrolides,lincosamides, streptogramins, althiomycins, oxazolidinones, nucleotideanalogs, thiostreptons (including micrococcin family), peptides,glutarimides, trichothecenes, TAN-1057, pleuromutilins, hygromycins,betacins, everninomicins, boxazomycins, and fusidanes.

Members of the chloramphenicol family include, for example,Chloramphenicol and Iodoamphenicol. Members of the macrolide familyinclude, for example, Biaxin (Clarithromycin), Zithromax (Azithromycin;azalide), Ketek (Telithromycin; ketolide), ABT-773, Tylosin, SpiramycinI, Spiramycin II, Spiramycin III, Erythromycin A, Carbomycin A,Telithromycin, Methymycin, Narbomycin, Lankamycin, Olcandomycin,Megalomycin, Chalcomycin, Niddamycin, Leucomycin, Angolamycin,Picromycin, and Relomycin. Members of the licosamide family include, forexample, Clindamycin and Lincomycin. Members of the streptogramin familyinclude, for example, Streptogramin A, Streptogramin B, Ostreogrycin G,Synercid, Virginiamycin S1, Virginiamycin S2, Virginiamycin S3,Virginiamycin S4, Vernamycin B, Vernamycin C, Patricin A, and PatricinB. A member of the althiomycin family, includes, for example,Althiomycin. Members of the oxazolidinone family, include, for example,Linezolid, Eperezolid, and DuP721. Members of the family of nucleotideanalogs include, for example, Sparsomycin, Puromycin, Anisomycin, andBlasticidin S. Members of the thiostrepton family include, for example,Thiostrepton, Siomycin, Sporangiomycin, Micrococcin M1, Micrococcin P,and Thiopeptin. Members of the peptide family include, for example,Viomycin, Capreomycin IA, Capreomycin IB, Capreomycin IIA, andCapreomycin IIB. Members of the glutarimide family include, for example,Cycloheximide, Streptovitacins, Streptimidone, Inactone, Actiphenol.Members of the trichothecene family include, for example, Trichodermin,Trichodermol, Trichodermone, Vomitoxin, T-2 toxin, Trichothecin,Nivalenol, and Verrucarin A. Tan-1057 includes Tan-1057A, Tan-1057B,Tan-1057C, and Tan-1057D. Pleuromutilins include, for example,Pleuromutilin, Tiamulin, Azamilin, and Valnemulin. Hygromycins includesthe Hygromycin A antibiotics. Betacins include, for example, the familyof betacin natural products and VCR4219. Everninomicins include, forexample, Ziracin, Avilamycin, Evernimicin, and Curamicin. Boxazomycinsinclude, for example, Boxazomycin A and B. Fusidanes include, forexample, fusidic acid and 17S, 20S-dihydrofusidic acid diethylene glycolhydrate

Inhibitors can be diffused into or soaked with the stabilized crystalsof the large ribosomal subunit as described in Examples 4 and 5 to forma complex with the large ribosomal subunit for collecting X-raydiffraction data. Alternatively, the inhibitors can be co-crystallizedwith the large ribosomal subunit by mixing the inhibitor with the largeribosomal subunit before precipitation with high salt.

Starting with the structure of the ribosome from H. Marismortui, thestructure of the ribosome from a non-targeted organism (for example, thehuman 60S ribosomal subunit) can be constructed by homology modeling,i.e., by changing the structure of residues at a target site of interestfor the residues at the same positions in of the non-target ribosome.This can be achieved by removing computationally the side chains fromthe ribosome of known structure and replacing them with the side chainsof the unknown structure put in sterically plausible positions. In thisway, it can be understood how the shapes of the target sites within thetargeted and non-targeted ribosomes differ. This process, therefore,provides information concerning how a molecule that binds the targetsite can be chemically altered in order to produce molecules that willbind tightly and specifically to the targeted ribosome but willsimultaneously be prevented from binding to the non-targeted ribosome.Likewise, knowledge of portions of the bound molecules that face thesolvent permit introduction of other functional groups for additionalpharmaceutical purposes. The process of homology structure modeling canalso be used to understand the mechanisms whereby mutant ribosomesbecome resistant to the effects of pharmaceuticals or pesticides, suchas herbicides or insecticides. Furthermore, with knowledge of theportions of the ribosomal subunit that participates in drug resistance,the skilled artisan may design new molecules that overcome the problemof drug resistance.

The use of homology structure modeling to design molecules that bindmore tightly to the target ribosome than to the non-target ribosome haswide-spread applicability. The methods outlined herein can be used tocontrol any targeted organism, for example, a pathogen, by designingmolecules that inhibit large ribosomal subunits of the targetedorganisms while failing to inhibit the 50S or 60S ribosomal subunit ofthe non-targeted organism, for example, a host, to the same extent ornot at all. The molecules identified or prepared by the methods of thepresent invention can be used to control the targeted organisms whilecausing the non-targeted organism little or no adverse effects. Thus,the molecules identified or developed using the methods of the presentinvention can be designed so that their administration kills the targetorganisms or inhibits some aspect of the biological functions of thetarget organisms while failing to have a similar effect on thenon-targeted organism. The adverse effects of the agent on the targetedorganisms may include, but are not limited to, death of the targetorganism; slowing growth rates; slowing or eliminating passage from onegrowth phase to another (e.g., extending the larval growth stage);slowing or eliminating reproduction, decreasing or preventing mating,decreasing or eliminating offspring production, limiting or eliminatingtarget organism weight gains; decreasing or eliminating feeding abilityand behaviors; and disrupting cellular, tissue and/or organ functions.

The novel agents contemplated by the present invention can be useful asherbicides, pesticides (e.g., insecticides, nematocides, rodenticides,etc.), miticides, or antimicrobial agents (e.g., antifungals,antibacterials, antiprotozoals, etc.) to target specific organisms. Forexample, the novel agents can target animal and plant parasiticnematodes, prokaryotic organisms (disease causing microbes), andeukaryotic multicellular pests. Specific examples of multicellular pestsinclude, but are not limited to, insects, fungi, bacteria, nematodes,mites and ticks, protozoan pathogens, animal-parasitic liver flukes, andthe like.

Herbicides, pesticides, miticides, and antimicrobial agents that inhibitprotein synthesis by interacting with ribosomes are known to the skilledartisan. A few examples are discussed below. These known agents can bemodified to obtain novel agents by using computer modeling techniquesand knowledge of the structure of ribosomes and ribosomal subunits andthe structure of ribosome/agent and ribosomal subunit/agent complexes.

The ketolide ABT-773 binds ribosomes tighter than erythromycin in S.pneumoniae and is able to defeat macrolide resistance in bacteria(Capobianco et al. (2000) Antimicrob. Agents Chemother. 44(6):1562-1567). The tools and methodologies of the present invention can beused to obtain erythromycin derivatives that bind the ribosomes orribosomal subunits of target bacteria more tightly than they bind theribosomes and ribosomal subunits of non-target animals. The targetbacteria can be any infectious bacteria, particularly S. pneumoniae, andeven more particularly erythromycin-resistant S. pneumoniae. Thenon-target animals can be any animal, particularly mammals, and evenmore particularly humans.

Examples of antibiotics that are inhibitors of protein synthesisinclude, but are not limited to, puromycin, cycloheximide,chloramphenicol, tetracycline, and streptomycin (Heldt (1996) PlantBiochemistry and Molecular Biology 21.2: 458-464). Puromycin, asdiscussed earlier, binds as an analogue of an aminoacyl-tRNA to theA-site and is added to nascent peptide chains, and, prevents furtherelongation steps in prokaryotes and eukaryotes. Cycloheximide inhibitspeptidyl transferase in eukaryotic ribosomes. Chloramphenicol inhibitspeptidyl transferase in prokaryotic ribosomes. Tetracycline binds to the30S subunit and inhibits the binding of aminoacyl-tRNA to prokaryoticribosomes much more than to eukaryotic ones. Streptomycin interacts with30S ribosomes which results in an incorrect recognition of mRNAsequences and thus inhibits initiation in prokaryotic ribosomes. U.S.Pat. No. 5,801,153 discloses antibiotics against pathogens.Aminoglycosides are examples of antibacterial antibiotics that appear toinhibit protein synthesis. However, there is a limitation to their usebecause of their ototoxic and nephrotoxic properties. Amikacin sulfate,Framycetin sulfate, Gentamycin sulfate, Kanamycin sulfate, Neomycinsulfate, Netilmicin sulfate, Paromomycin sulfate, Sissomycin sulfate,Tobramycin, Vancomycin hydrochloride, and Viomycin sulfate are themembers of the aminoglycoside family. The tools and methodologies of thepresent invention can be used to obtain derivatives of any antibiotic ofchoice so that they inhibit the protein synthesis of target organisms toa greater degree than they inhibit the protein synthesis of non-targetorganisms, such as humans.

Examples of targeted and non-targeted organisms include, but are notlimited to, those provided in Table 21.

TABLE 21 Examples of Classes of Molecules which can be Identified and/orDeveloped by the Methods of the Invention and ApplicableTarget/Non-Target Organisms. Non-Target Type of Molecule TargetOrganisms Organisms Herbicides Dicotyledonous plants Monocotyledonousplants Herbicides Grasses Soybeans, potatoes, coffee Insecticides Flies,Mites Honey bees Pesticides Ticks Deer Pesticides Lice Birds MiticidesParasitic mites (mange) Dogs Antimicrobial Agents Streptococcuspneumoniae Humans (Antibacterials) Antimicrobial Agents Clostridiumdfficile Escherichia coli (Antibacterials) Antimicrobial Agents Erysiphegraminis Barley (Antifungals) Antimicrobial Agents Toxoplasma gondiiAnimals (Antiprotozoals) Poisons Rats Dogs, cats, (Rodenicides) humans

It is contemplated that the tools and methodologies of the presentinvention can be used to obtain inhibitors of protein synthesis oftarget insects, such as bollworms and mosquitoes, more than they inhibitthe protein synthesis of non-target insects, such as beetles of thefamily Coccinellidae (e.g., ladybugs) and Apis mellifera (honey bees).Other possible target insects include, but are not limited to, insectsselected from the orders Coleoptera (beetles), Diptera (flies,mosquitoes), Hymenoptera (wasps, ants, sawflies), Lepidoptera(butterflies and moths), Mallophaga (lice), Homoptera (whiteflies,aphids), Hemiptera (bugs), Orthroptera (locusts, cockroaches),Thysanoptera (thrips), Dermaptera (earwigs), Isoptera, Anoplura,Siphonaptera, and Trichoptera (caddis flies).

Furthermore, it is contemplated that the tools and methodologies of thepresent invention can be used to obtain inhibitors of protein synthesisof target plants which inhibit protein synthesis of the target plantsmore than they inhibit the protein synthesis of non-target plants andanimals. The target plants can be any unwanted plant species, particularweeds, and even more particularly noxious weeds. Whether or not aparticular plant is considered a weed will depend upon the context inwhich it is growing. For example, unwanted Zea mays (corn) plantsgrowing in a Glycine max (soybean) field could be considered unwantedweeds. Examples of weeds which are likely target plants include, but arenot limited to, Allium vineale (wild garlic), Bromus tectorum (downybrome), Triticum cylindricum (jointed goatgrass), Amaranthus spp.(pigsweed), Chenopodium album (lambsquarters), Avena fatua (wild oats),B. secalinus (cheat), Echinochloa crus-galli (barnyardgrass), Alopecurusmyosuroides (blackgrass), Setaria faberii (giant foxtail), Xanthiumstrumarium (common cocklebur), Ambrosia artemisiifolia (common ragweed),and Ipomoea spp. (morning glories). The non-target organisms can be anyplant, particularly any desirable plant, and even more particularly anycrop plant. The non-target organisms can also be any animals,particularly mammals, and even more particularly humans. In onepreferred embodiment, the tools and methodologies of the presentinvention can be used to produce protein synthesis inhibitors which killor injure one or more noxious weed species but fail to harm non-targetplants and animals.

Target bacteria of interest include, but are not limited to,Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus bovis, Streptococcus pneumoniae, Moraxella catarrhalis,Neisseria gonorrhoeae, Neisseria meningitides, Bacillus anthracis,Corynebacterium diphtheriae, Listeria monocytogenes, Erysipelothrixrhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridiumdifficile, Eschericia coli, Proteus mirabilis, Psuedomonas aeruginosa,Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus ducreyi,Yersinia pestis, Yersinia enterocolitica, Francisella tularensis,Pasteurella multocida, Vibrio cholerae, Flavobacterium meningosepticum,Pseudomonas mallei, Pseudomonas pseudomallei, Campylobacter jejuni,Campylobacter fetus, Fusobacterium nucleatum, Calyminmatobacteriumgranulomatis, Streptobacillus moniliformis, Legionella pneumophila,Mycobacterium avium-intracellulare, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Treponeina pertenue, Borreliaburgdorferi, Borrelia recurrentis, Actinomyces isrealii, Nocardiaasteroides, Ureaplasma urealyticum, Mycoplasma pneumoniae, Chlamydiapsittaci, Chlamydia trachomatis, Chlamydia pnemoniae, Pneumocystiscarinii, Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii,Cryplococcus neoformans.

Once a candidate molecule has been designed or selected by the abovemethods, the affinity with which that molecule may bind to the ribosomeor ribosomal subunit may be tested and optimized by computationalevaluation and/or by testing biological activity after synthesizing thecompound. Candidate molecules may interact with the ribosomes orribosomal subunits in more than one conformation each of which has asimilar overall binding energy. In those cases, the deformation energyof binding may be considered to be the difference between the energy ofthe free molecule and the average energy of the conformations observedwhen the molecule binds to the ribosomes or ribosomal subunits, morepreferably to the large ribosomal subunits, and even more preferably tothe 50S ribosomal subunits.

A molecule designed or selected as binding to a ribosome or ribosomalsubunit may be further computationally optimized so that in its boundstate it preferably lacks repulsive electrostatic interaction with thetarget region. Such non-complementary (e.g., electrostatic) interactionsinclude repulsive charge-charge, dipole-dipole and charge-dipoleinteractions. Specifically, the sum of all electrostatic interactionsbetween the inhibitor and the enzyme when the inhibitor is bound to theribosome or the ribosomal subunit, preferably make a neutral orfavorable contribution to the enthalpy of binding. Weak bindingcompounds can also be designed by these methods so as to determine SAR.

Specific computer programs that can evaluate a compound deformationenergy and electrostatic interaction arc available in the art. Examplesof suitable programs include: Gaussian 92, revision C (M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa.); AMBER, version 4.0 (P. A. Kollman,University of California at San Francisco, Calif.); QUANTA/CHARMM(Molecular Simulations, Inc., Burlington, Mass.); OPLS-AA (“OPLS ForceFields.” W. L. Jorgensen. Encyclopedia of Computational Chemistry,Schleyer, Ed.; Wiley: New York, 1998; Vol. 3, pp 1986-1989.) and InsightII/Discover (Biosysm Technologies Inc., San Diego, Calif.). Theseprograms may be implemented, for instance, using a Silicon Graphicsworkstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Otherhardware systems and software packages are known to those skilled in theart.

Once a molecule of interest has been selected or designed, as describedabove, substitutions may then be made in some of its atoms or sidegroups in order to improve or modify its binding properties. Generally,initial substitutions are conservative, i.e., the replacement group willapproximate the same size, shape, hydrophobicity and charge as theoriginal group. It should, of course, be understood that componentsknown in the art to alter conformation should be avoided. Suchsubstituted chemical compounds may then be analyzed for efficiency offit to the ribosome or ribosomal subunit by the same computer methodsdescribed in detail, above.

In addition, the actual ribosome-related ligands, complexes or mimeticsmay be crystallized and analyzed using X-ray diffraction. Thediffraction pattern co-ordinates are similarly used to calculate thethree-dimensional interaction of a ligand and the ribosome, ribosomalsubunit, or a mimetic, in order to confirm that the ligand binds to, orchanges the conformation of, a particular site on the ribosome orribosomal subunit, or where the mimetic has a similar three-dimensionalstructure to that of a ribosome, ribosomal subunit or a fragmentthereof.

3. Synthesis of Lead Molecules

A lead molecule of the present invention can be, but is not limited to,at least one selected from a lipid, nucleic acid, peptide, small organicor inorganic molecule, chemical compound, element, saccharide, isotope,carbohydrate, imaging agent, lipoprotein, glycoprotcin, enzyme,analytical probe, and an antibody or fragment thereof, any combinationof any of the foregoing, and any chemical modification or variant of anyof the foregoing. In addition, a lead molecule may optionally comprise adetectable label. Such labels include, but are not limited to, enzymaticlabels, radioisotope or radioactive compounds or elements, fluorescentcompounds or metals, chemiluminescent compounds and bioluminescentcompounds. Well known methods may be used for attaching such adetectable label to a lead molecule.

Methods useful for synthesizing lead molecules such as lipids, nucleicacids, peptides, small organic or inorganic molecules, chemicalcompounds, elements, saccharides, isotopes, carbohydrates, imagingagents, lipoproteins, glycoproteins, enzymes, analytical probes,antibodies, and antibody fragments are well known in the art. Suchmethods include the traditional approach of synthesizing one such leadmolecule, such as a single defined peptide, at a time, as well ascombined synthesis of multiple lead molecules in a one or morecontainers. Such multiple lead molecules may include one or morevariants of a previously identified lead molecule. Methods for combinedsynthesis of multiple lead molecules are particularly useful inpreparing combinatorial libraries, which may be used in screeningtechniques known in the art.

By way of example, it is well known in the art that multiple peptidesand oligonucleotides may be simultaneously synthesized. Lead moleculesthat are small peptides up to 50 amino acids in length, may besynthesized using standard solid-phase peptide synthesis procedures, forexample, procedures similar to those described in Merrifield (1963) J.Am. Chem. Soc., 85: 2149. For example, during synthesis, N-α-protectedamino acids having protected side chains are added stepwise to a growingpolypeptide chain linked by its C-terminal end to an insoluble polymericsupport, e.g., polystyrene beads. The peptides are synthesized bylinking an amino group of an N-α-deprotected amino acid to an α-carboxygroup of an N-α-protected amino acid that has been activated by reactingit with a reagent such as dicyclohexylcarbodiimide. The attachment of afree amino group to the activated carboxyl leads to peptide bondformation. The most commonly used N-α-protecting groups include Bocwhich is acid labile and Fmoc which is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. Then, the N-α-protecting group is removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arecleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides, for example greater than about 50amino acids in length, typically are derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein. See for example,Atherton et al. (1963) Solid Phase Peptide Synthesis: A PracticalApproach (IRL Press), and Bodanszky (1993) Peptide Chemistry, APractical Textbook, 2nd Ed. Springer-Verlag, and Fields et al. (1990)Int. J. Peptide Protein Res. 35:161-214.

Purification of the resulting peptide is accomplished using conventionalprocedures, such as preparative HPLC, e.g., gel permeation, partitionand/or ion exchange chromatography. The choice of appropriate matricesand buffers are well known in the art and so are not described in detailherein.

It is contemplated that a synthetic peptide in accordance with theinvention may comprise naturally occurring amino acids, unnatural aminoacids, and/or amino acids having specific characteristics, such as, forexample, amino acids that are positively charged, negatively charged,hydrophobic, hydrophilic, or aromatic. As used herein, the term“naturally occurring amino acids” refers to the L-isomers of amino acidsnormally found in proteins. The predominant naturally occurring aminoacids are glycine, alanine, valine, leucine, isoleucine, serine,methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine,proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine,arginine, and lysine. Unless specifically indicated, all amino acids arereferred to in this application are in the L-form. Furthermore, as usedherein, the term “unnatural amino acids” refers to amino acids that arenot naturally found in proteins. For example, selenomethionine.

Amino acids that are “positively charged” include any amino acid havinga positively charged side chain under normal physiological conditions.Examples of positively charged naturally occurring amino acids include,for example, arginine, lysine, and histidine. Conversely, amino acidsthat are “negatively charged” include any amino acid having a negativelycharged side chains under normal physiological conditions. Examples ofnegatively charged naturally occurring amino acids include, for example,aspartic acid and glutamic acid.

As used herein, the term “hydrophobic amino acid” includes any aminoacids having an uncharged, nonpolar side chain that is relativelyinsoluble in water. Examples of naturally occurring hydrophobic aminoacids include, for example, alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. In addition, as usedherein, the term “hydrophilic amino acid” refers to any amino acidshaving an uncharged, polar side chain that is relatively soluble inwater. Examples of naturally occurring hydrophilic amino acids include,for example, serine, threonine, tyrosine, asparagine, glutamine andcysteine.

Finally, as used herein, the term “aromatic” refers to amino acidresidues which side chains have delocalized conjugated system. Examplesof aromatic residues include, for example, phenylalanine, tryptophan,and tyrosine.

With regard to the production of non-peptide small organic moleculeswhich act as a ligand in the present invention, these molecules can besynthesized using standard organic chemistries well known and thoroughlydocumented in the patent and other literatures.

Many of the known methods useful in synthesizing lead of the presentinvention may be automated, or may otherwise be practiced on acommercial scale. As such, once a lead molecule has been identified ashaving commercial potential, mass quantities of that molecule may easilybe produced.

4. Characterization of Molecules

Molecules designed, selected and/or optimized by methods describedabove, once produced, may be characterized using a variety of assaysknown to those skilled in the art to determine whether the compoundshave biological activity. For example, the molecules may becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening may be used to speed up analysisusing such assays. As a result, it may be possible to rapidly screen newmolecules for their ability to interact with a ribosome or ribosomalsubunit using the tools and methods of the present invention. Generalmethodologies for performing high-throughput screening are described,for example, in Devlin (1998) High Throughput Screening, Marcel Dekker;and U.S. Pat. No. 5,763,263. High-throughput assays can use one or moredifferent assay techniques including, but not limited to, thosedescribed below.

(1) Surface Binding Studies. A variety of binding assays may be usefulin screening new molecules for their binding activity. One approachincludes surface plasmon resonance (SPR) which can be used to evaluatethe binding properties molecules of interest with respect to a ribosome,ribosomal subunit or a fragment thereof.

SPR methodologies measure the interaction between two or moremacromolecules in real-time through the generation of aquantum-mechanical surface plasmon. One device, (BlAcore Biosensor RTMfrom Pharmacia Biosensor, Piscatawy, N.J.) provides a focused beam ofpolychromatic light to the interface between a gold film (provided as adisposable biosensor “chip”) and a buffer compartment that can beregulated by the user. A 100 nm thick “hydrogel” composed ofcarboxylated dextran which provides a matrix for the covalentimmobilization of analytes of interest is attached to the gold film.When the focused light interacts with the free electron cloud of thegold film, plasmon resonance is enhanced. The resulting reflected lightis spectrally depleted in wavelengths that optimally evolved theresonance. By separating the reflected polychromatic light into itscomponent wavelengths (by means of a prism), and determining thefrequencies which are depleted, the BIAcore establishes an opticalinterface which accurately reports the behavior of the generated surfaceplasmon resonance. When designed as above, the plasmon resonance (andthus the depletion spectrum) is sensitive to mass in the evanescentfield (which corresponds roughly to the thickness of the hydrogel). Ifone component of an interacting pair is immobilized to the hydrogel, andthe interacting partner is provided through the buffer compartment, theinteraction between the two components can be measured in real timebased on the accumulation of mass in the evanescent field and itscorresponding effects of the plasmon resonance as measured by thedepletion spectrum. This system permits rapid and sensitive real-timemeasurement of the molecular interactions without the need to labeleither component.

(2) Immunodiagnostics and Immunoassay. These are a group of techniquesthat can be used for the measurement of specific biochemical substances,commonly at low concentrations in complex mixtures such as biologicalfluids, that depend upon the specificity and high affinity shown bysuitably prepared and selected antibodies for their complementaryantigens. A substance to be measured must, of necessity, beantigenic—either an immunogenic macromolecule or a haptenic smallmolecule. To each sample a known, limited amount of specific antibody isadded and the fraction of the antigen combining with it, often expressedas the bound:free ratio, is estimated, using as indicator a form of theantigen labeled with radioisotope (radioimmunoassay), fluorescentmolecule (fluoroimmunoassay), stable free radical (spin immunoassay),enzyme (enzyme immunoassay), or other readily distinguishable label.

Antibodies can be labeled in various ways, including: enzyme-linkedimmunosorbent assay (ELISA); radioimmuno assay (RIA); fluorescentimmunoassay (FIA); chemiluminescent immunoassay (CLIA); and labeling theantibody with colloidal gold particles (immunogold).

Common assay formats include the sandwich assay, competitive orcompetition assay, latex agglutination assay, homogeneous assay,microtitre plate format and the microparticle-based assay.

(3) Enzyme-linked immunosorbent assay (ELISA). ELISA is animmunochemical technique that avoids the hazards of radiochemicals andthe expense of fluorescence detection systems. Instead, the assay usesenzymes as indicators. ELISA is a form of quantitative immunoassay basedon the use of antibodies (or antigens) that are linked to an insolublecarrier surface, which is then used to “capture” the relevant antigen(or antibody) in the test solution. The antigen-antibody complex is thendetected by measuring the activity of an appropriate enzyme that hadpreviously been covalently attached to the antigen (or antibody).General methods and compositions for practicing ELISA are described, forexample, in Crowther (1995) ELISA—Theory and Practice (Methods inMolecular Biology), Humana Press; Challacombe and Kemeny, (1998) ELISAand Other Solid Phase Immunoassays—Theoretical and Practical Aspects,John Wiley; Kemeny, (1991) A Practical Guide to ELISA, Pergamon Press;Ishikawa, (1991) Ultrasensitive and Rapid Enzyme Immunoassay (LaboratoryTechniques in Biochemistry and Molecular Biology) Elsevier.

(4) Colorimetric Assays. Colorimetry is any method of quantitativechemical analysis in which the concentration or amount of a compound isdetermined by comparing the color produced by the reaction of a reagentwith both standard and test amounts of the compound, often using acolorimeter. A calorimeter is a device for measuring color intensity ordifferences in color intensity, either visually or photoelectrically.

Standard calorimetric assays of beta-galactosidase enzymatic activityare well known to those skilled in the art (see, for example, Norton etal. (1985) Mol. Cell. Biol. 5: 281-290). A calorimetric assay can beperformed on whole cell lysates usingO-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma) as the substrate in astandard colorimetric beta-galactosidase assay (Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress). Automated colorimetric assays are also available for thedetection of β-galactosidase activity, as described in U.S. Pat. No.5,733,720.

(5) Immunofluorescence Assays. Immunofluorescence or immunofluorescencemicroscopy is a technique in which an antigen or antibody is madefluorescent by conjugation to a fluorescent dye and then allowed toreact with the complementary antibody or antigen in a tissue section orsmear. The location of the antigen or antibody can then be determined byobserving the fluorescence by microscopy under ultraviolet light.

A general description of immunofluorescent techniques appears forexample, in Knapp et al. (1978) Immunofluorescence and Related StainingTechniques, Elsevier; Allan, (1999) Protein Localization by FluorescentMicroscopy—A Practical Approach (The Practical Approach Series) OxfordUniversity Press; Caul, (1993) Immunofluorescence Antigen DetectionTechniques in Diagnostic Microbiology, Cambridge University Press. Fordetailed explanations of immunofluorescent techniques applicable to thepresent invention, see, for example, U.S. Pat. Nos. 5,912,176;5,869,264; 5,866,319; and 5,861,259.

(6) Fluorescence Polarization. Fluorescence polarization (FP) is ameasurement technique that can readily be applied to protein-protein andprotein-ligand interactions in order to derive IC₅₀s and Kds of theassociation reaction between two molecules. In this technique one of themolecules of interest is conjugated with a fluorophore. This isgenerally the smaller molecule in the system (in this case, the moleculeof interest). The sample mixture, containing both the ligand-probeconjugate and the ribosome, ribosomal subunit or fragment thereof, isexcited with vertically polarized light. Light is absorbed by the probefluorophores, and re-emitted a short time later. The degree ofpolarization of the emitted light is measured. Polarization of theemitted light is dependent on several factors, but most importantly onviscosity of the solution and on the apparent molecular weight of thefluorophore. With proper controls, changes in the degree of polarizationof the emitted light depends only on changes in the apparent molecularweight of the fluorophore, which in-turn depends on whether theprobe-ligand conjugate is free in solution, or is bound to a receptor.Binding assays based on FP have a number of important advantages,including the measurement of IC₅₀s and Kds under true homogenousequilibrium conditions, speed of analysis and amenity to automation, andability to screen in cloudy suspensions and colored solutions.

(7) Protein Synthesis. It is contemplated that, in addition tocharacterization by the foregoing biochemical assays, the molecule ofinterest may also be characterized as a modulator (for example, aninducer of protein synthesis or an inhibitor of protein synthesis) ofthe functional activity of the ribosome or ribosomal subunit.

Inhibitors of protein synthesis may be assayed on the cellular level.For example, molecules of interest can be assayed for inhibitory actionagainst organisms, for example, micro-organism, by growing themicro-organism of interest in media either containing or lacking themolecule of interest. Growth inhibition may be indicative that themolecule may be acting as a protein synthesis inhibitor.

Furthermore, more specific protein synthesis inhibition assays may beperformed by administering the compound to a whole organism, tissue,organ, organelle, cell, a cellular or subcellular extract, or a purifiedribosome preparation and observing its pharmacological and inhibitoryproperties by determining, for example, its inhibition constant (IC₅₀)for inhibiting protein synthesis. Incorporation of ³H leucine or ³⁵Smethionine, or similar experiments can be performed to investigateprotein synthesis activity.

A change in the amount or the rate of protein synthesis in the cell inthe presence of a molecule of interest indicates that the molecule is aninducer of protein synthesis. A decrease in the rate or the amount ofprotein synthesis indicates that the molecule is a inhibitor of proteinsynthesis.

In addition, the antibacterial activity of the compounds of the presentinvention against bacterial pathogens can be demonstrated by thecompound's ability to inhibit growth of defined strains of humanpathogens. For this purpose, a panel of bacterial strains can beassembled to include a variety of target pathogenic species, somecontaining resistance mechanisms that have been characterized. Use ofsuch a panel of organisms permits the determination ofstructure-activity relationships not only in regards to potency andspectrum, but also with a view to obviating resistance mechanisms. Theassays may be performed in microtiter trays according to conventionalmethodologies as published by The National Committee for ClinicalLaboratory Standards (NCCLS) guidelines (NCCLS. M7-A5-Methods forDilution Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically; Approved Standard-Fifth Edition. NCCLS Document M100-S12/M7(ISBN 1-56238-394-9).

H. Drug Formulation and Administration

It is contemplated that once identified, the active molecules of theinvention may be incorporated into any suitable carrier prior to use.More specifically, the dose of active molecule, mode of administrationand use of suitable carrier will depend upon the target and non-targetorganism of interest.

It is contemplated that with regard to mammalian recipients, thecompounds of interest may be administered by any conventional approachknown and/or used in the art. Thus, as appropriate, administration canbe oral or parenteral, including intravenous and intraperitoneal routesof administration. In addition, administration can be by periodicinjections of a bolus, or can be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an intrvenous bag). In certain embodiments, the compounds of theinvention can be therapeutic-grade. That is, certain embodiments complywith standards of purity and quality control required for administrationto humans. Veterinary applications are also within the intended meaningas used herein.

The formulations, both for veterinary and for human medical use, of thedrugs according to the present invention typically include such drugs inassociation with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredient(s). The carrier(s) should be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is known in theart. Except insofar as any conventional media or agent is incompatiblewith the active compound, use thereof in the compositions iscontemplated. Supplementary active compounds (identified or designedaccording to the invention and/or known in the art) also can beincorporated into the compositions. The formulations may conveniently bepresented in dosage unit form and may be prepared by any of the methodswell known in the art of pharmacy/microbiology. In general, someformulations are prepared by bringing the drug into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

A pharmaceutical composition of the invention should be formulated to becompatible with its intended route of administration. Examples of routesof administration include oral or parenteral, e.g., intravenous,intradermal, inhalation, transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., (1990). Formulations for parenteral administration can alsoinclude glycocholate for buccal administration, methoxysalicylate forrectal administration, or cutric acid for vaginal administration. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Suppositories forrectal administration also can be prepared by mixing the drug with anon-irritating excipient such as cocoa butter, other glycerides, orother compositions which are solid at room temperature and liquid atbody temperatures. Formulations also can include, for example,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes, and the like. Formulations for directadministration can include glycerol and other compositions of highviscosity. Other potentially useful parenteral carriers for these drugsinclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationadministration can contain as excipients, for example, lactose, or canbe aqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Retention enemas also can be used for rectal delivery.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units such as capsules, gelatin capsules,sachets, tablets, troches, or lozenges, each containing a predeterminedamount of the drug; in the form of a powder or granules; in the form ofa solution or a suspension in an aqueous liquid or non-aqueous liquid;or in the form of an oil-in-water emulsion or a water-in-oil emulsion.The drug may also be administered in the form of a bolus, electuary orpaste. A tablet may be made by compressing or moulding the drugoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the drug in afree-flowing form such as a powder or granules, optionally mixed by abinder, lubricant, inert diluent, surface active or dispersing agent.Moulded tablets may be made by moulding, in a suitable machine, amixture of the powdered drug and suitable carrier moistened with aninert liquid diluent.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration may be in theform of a sterile aqueous preparation of the drug which may be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems may also be used to present the drug for bothintra-articular and ophthalmic administration.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pasts; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the drug with a dermatologically acceptablecarrier such as a lotion, cream, ointment or soap. Particularly usefulare carriers capable of forming a film or layer over the skin tolocalize application and inhibit removal. For topical administration tointernal tissue surfaces, the agent can be dispersed in a liquid tissueadhesive or other substance known to enhance adsorption to a tissuesurface. For example, hydroxypropylcellulose or fibrinogen/thrombinsolutions can be used to advantage. Alternatively, tissue-coatingsolutions, such as pectin-containing formulations can be used.

For inhalation treatments, inhalation of powder (self-propelling orspray formulations) dispensed with a spray can, a nebulizer, or anatomizer can be used. Such formulations can be in the form of a finepowder for pulmonary administration from a powder inhalation device orself-propelling powder-dispensing formulations. In the case ofself-propelling solution and spray formulations, the effect may beachieved either by choice of a valve having the desired spraycharacteristics (i.e., being capable of producing a spray having thedesired particle size) or by incorporating the active ingredient as asuspended powder in controlled particle size. For administration byinhalation, the compounds also can be delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfilsidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds typically areformulated into ointments, salves, gels, or creams as generally known inthe art.

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialsalso can be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.

Oral or parenteral compositions can be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

As noted above, drugs identified or designed according to the inventioncan be formulated into pharmaceutical compositions by admixture withpharmaceutically acceptable nontoxic excipients and carriers. Suchcompositions can be prepared for parenteral administration, particularlyin the form of liquid solutions or suspensions; for oral administration,particularly in the form of tablets or capsules; or intranasally,particularly in the form of powders, nasal drops or aerosols. Whereadhesion to a tissue surface is desired the composition can include thedrug dispersed in a fibrinogen-thrombin composition or otherbioadhesive. The drug then can be painted, sprayed or otherwise appliedto the desired tissue surface. Alternatively, the drugs can beformulated for parenteral or oral administration to humans or othermammals, for example, in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the drug to targettissue for a time sufficient to induce the desired effect.

Where the active compound is to be used as part of a transplantprocedure, it can be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. Thedrug can be provided to the donor host. Alternatively or, in addition,once removed from the donor, the organ or living tissue can be placed ina preservation solution containing the active compound. In all cases,the active compound can be administered directly to the desired tissue,as by injection to the tissue, or it can be provided systemically,either by oral or parenteral administration, using any of the methodsand formulations described herein and/or known in the art.

Where the drug comprises part of a tissue or organ preservationsolution, any commercially available preservation solution can be usedto advantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution.

The effective concentration of the compounds to be delivered in atherapeutic composition will vary depending upon a number of factors,including the final desired dosage of the compound to be administeredand the route of administration. The preferred dosage to be administeredalso is likely to depend on such variables as the type and extent ofdisease or indication to be treated, the overall health status of theparticular patient, the relative biological efficacy of the compounddelivered, the formulation of the drug, the presence and types ofexcipients in the formulation, and the route of administration. Ingeneral terms, the drugs of this invention can be provided to anindividual using typical dose units deduced from the earlier-describedmammalian studies using non-human primates and rodents.

When the active compounds are nucleic acid molecules, the nucleic acidmay be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see, U.S. Pat. No.5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

When an active compound of the invention is intended for administrationto a plant host, the invention may be applied directly to the plantenvironment, for example, to the surface of leaves, buds, roots orfloral parts. Alternatively, the present invention can be used as a seedcoating. The determination of an effective amount of the presentinvention as required for a particular plant is within the skill of theart and will depend on such factors as the plant species, method ofplanting, and soil type. It is contemplated that compositions containingdrugs of the invention can be prepared by formulating such drugs withadjuvants, diluents, carriers, etc., to provide compositions in the formof filings/divided particulate solids, granules, pellets, wetablepowders, dust, aqueous suspensions or dispersions, and emulsions. It isfurther contemplated to use such drugs in capsulated form, for example,the drugs can be encapsulated within polymer, gelatin, lipids or otherformulation aids such as emulsifiers, surfactants wetting agents,antifoam agents and anti-freeze agents, may be incorporated into suchcompositions especially if such compositions will be stored for anyperiod of time prior to use. Application of compositions containingdrugs of the invention as the active agent can be carried out byconventional techniques. When an active compound is intended foradministration to an insect host, standard methods such as, but notlimited to, aerial dispersal are contemplated.

Active compound identified or designed by a method of the invention alsoinclude precursors of the active compounds. The term precursors refersto a pharmacologically inactive (or partially inactive) derivative of aparent molecule that requires biotransformation, either spontaneous orenzymatic, within the organism to release the active compounds.Precursors are variations or derivatives of the compounds of theinvention which have groups cleavable under metabolic conditions.Precursors become the active compounds of the invention which arepharmaceutically active in vivo, when they undergo solvolysis underphysiological conditions or undergo enzymatic degradation. Precursorforms often offer advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see Bundgard, Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, TheOrganic Chemistry of Drug Design and Drug Action, pp. 352-401, AcademicPress, San Diego, Calif. (1992).

Active compound as identified or designed by the methods describedherein can be administered to individuals to treat disorders(prophylactically or therapeutically). In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

With regard to mammals, it is contemplated that the effective dose of aprotein synthesis inducer or inhibitor will be in the range of about0.01 to about 50 mg/kg, preferably about 0.1 to about 10 mg/kg of bodyweight, administered in single or multiple doses. Typically, the induceror inhibitor may be administered to a human recipient in need oftreatment at a daily dose range of about 1 to about 2000 mg per patient.

In light of the foregoing general discussion, the specific examplespresented below are illustrative only and are not intended to limit thescope of the invention. Other generic and specific configurations willbe apparent to those persons skilled in the art.

III. EXAMPLES A. Example 1 Preparation of 50S Ribosomal Subunit Crystals

H. Marismortui (ATCC 43049) was grown as described previously (Ban etal. (1998) supra) on a slightly modified version of ATCC culture medium1230, which was supplemented with 4.3 g of yeast extract, 5.1 g of Tris,and 3.4 g of glucose per liter. Bacteria were grown at 37° C. to anOD_(550 nm) between 1.0 and 2.2. They were harvested by centrifugation,and stored at −80° C. until use. Cells were ruptured using a Frenchpress. Ribosomes were prepared from lysates by centrifugation, andsubunits were isolated on sucrose gradients as previously described(Shevack et al. (1985) FEBS Lett. 184: 68-71).

The crystals were prepared and stabilized as follows:

1. Reverse Extraction

-   (1) Mix 1 mg of subunits in a concentrated 50S ribosomal subunit    stock (30 mg/ml in 1.2 M KCl, 0.5 M NH₄Cl, 20 mM MgCl₂, 10 mM Tris,    1 mM CdCl₂, 5 mM Tris, pH 7.5) with ½ volume of 30% PEG6000 (300 g    PEG, 700 ml H₂O to make 1 liter of 30% PEG; filter through 0.2 μm    filter). Leave on ice for 1 to 2 hours.-   (2) Spin down the precipitate in an eppendorf tube for about 30    seconds using a desktop centrifuge.-   (3) Remove the supernatant and add 100 μl of RE-buffer (7% PEG6000,    1.2 M KCl, 0.5 M NH₄Cl, 100 mM KAc, 30 mM MgCl₂, 10 mM Tris, 10 mM    MES (pH 7.5), and 1 mM CdCl₂)-   (4) Resuspend the pellet at room temperature by mixing with a P200    pipette set at 50 μl. The resuspended material should appear a    little cloudy.-   (5) Wrap the eppendorf tube in aluminum foil and leave for    equilibration at room temperature for 30-60 min. The solution will    be saturated with 50S ribosomal subunits.-   (6) Spin down the precipate for 2 minutes in desk-top centrifuge at    room temperature, and transfer the supernatant to new eppendorf    tube. A little pellet should be found in the tube used for    centrifugation. Store the supernatant at room temperature.-   (7) Place 8-10 μl of the supernatant in the sample well of a sitting    drop tray (Charles-Supper). Streak seed one hour later from a seed    stock. Seed stock is prepared by putting previously grown crystals    in stabilizing solution buffer A (see below), and then vortexing    them violently. To streak seed, a human hair cleaned with water and    ethanol and then dried is passed through the vortexed solution and    then touched on the new crystallization drop. Drops should look    cloudy. The reservoirs in the sitting drop trays contain 1000 μl of    a solution containing 8% PEG6000, 1.2 M KCl, 0.5 M NH₄Cl, 100 mM    KAc, 6.5 mM HAc (yields pH 5.8), 30 mM MgCl₂, and 1 mM CdCl₂.-   (8) After one day, see if the seeding is successful and, if so, let    the crystals grow for three weeks.

2. Stabilization Protocol

When the crystals have finished growing (after approximately 3 weeks),open each sitting drop chamber by making just a single cut (slit) goingfrom the middle to the edge of the well. Through this narrow slit, addto each drop in each reservoir 10 μl of buffer A (1.2 M KCl, 0.5 MNH₄Cl, 30 mM MgCl₂, 10% PEG6000, 1 mM CdCl₂, 100 mM KAc, 10 mM Tris(titrated to final pH 6.1), 30 mM MES) at room temperature and 45 μl ofBuffer C (0.667 M MES, 0.333 M Tris).

Place trays in a plastic box with a lid, and place in a 16° C. incubatorfor approximately one day, and then lower the temperature of theincubator to 12° C. for another day. Then place the plastic box in apolystyrene container with a lid, and put in a cold room for yet anotherday. Crystals can be stored like this for a long time, but need toundergo a further buffer change prior to use.

Make the following transition series using buffer A (see above) andbuffer B (1.7 M NaCl, 0.5 M NH₄Cl, 30 mM MgCl₂, 1 mM CdCl₂, 12% PEG6000,20% EG, 100 mM KAC (titrated to final pH 5.8 with HAC) to give finalratios of buffer B to buffer A of: 1/16, ⅛, ¼, ½, ¾. All solutionsshould be kept at cold room temperature. All of the followingmanipulations of the drops will take place through the narrow slit.

-   (1) Add 40 μl “ 1/16” to the drop, and leave for 15 minutes.-   (2) Add 40 μl “⅛” to the drop, and leave for 30-60 minutes.-   (3) Take out 40 μl from the drop (and discard it in the reservoir),    add 40 μl “¼”, and leave for 30-60 minutes.-   (4) Take out 40 μl from the drop (and discard it in the reservoir),    add 40 μl “½”, and lea for 15 minutes.-   (5) Take out 40 μl from the drop (and discard it in the reservoir),    add 40 μl “¾”, and lea for 15 minutes.-   (6) Take out 40 μl from the drop (and discard it in the reservoir),    add 40 μl buffer B, and leave for 15 minutes.-   (7) Take out 60-80 μl from the drop (and discard it in the    reservoir), add 60-80 μl Buffer B, and replace reservoirs with 500    μl buffer B.

B. Example 2 Determination of the Crystal Structure of the 50S RibosomalSubunit, with the Initial Refinement

All data, except the two native data sets, were collected at theNational Synchrotron Light Source (Brookhaven) from crystals frozen at100 K, using beamlines X12b and X25 and recorded using a 345 mm MARimaging plate. For each heavy atom derivative, anomalous diffractiondata were collected at the wavelength corresponding to the peakanomalous scattering. The beam size was 100×100 μm for most datacollections at X25 and 200×200 μm at beamline X12b. The crystals werealigned along the long axis of the unit cell (570 Å) so that 1.0°oscillations could be used to collect reflections out to a maximum of2.7 Å resolution at the edge of the MAR detector. At beamline X12b thecrystal to detector distances varied between 450.0 mm and 550.0 mmdepending on wavelength, crystal quality, and beam divergence, and itwas chosen so that maximum resolution data could be collected whileavoiding overlapping of spots. At beamline X25 the detector waspositioned on a rigid platform at 480 mm which allowed data collectionto 3.2 Å for iridium and osmium derivatives with the wavelength set atthe anomalous edge. Native data to 2.4 Å resolution were collected atthe structural biology beamline ID19 of the Advanced Photon Source(Argonnc) using a CCD detector. Data sets were processed by using DENZOand SCALEPACK (Otwinowski, (1993) Data Collection and Processing).

Heavy atom based phasing was extended to 3.2 Å resolution by combiningMIR phases calculated for two different isomorphous groups of data (MIR1and MIR2, Table 1) with single derivative anomalous dispersion (SAD)phases. The best two derivatives were osmium pentamine and iridiumhexamine, each of which contained a large number of binding sites (Table1). Several other derivatives with smaller number of sites furtherimproved map quality. All phasing was done by maximum likelihood methodimplemented in CNS (Brünger et al. (1998) supra) with the exception ofthe Ta₆Br₁₂ derivative, which was refined in SHARP (de La Fortelle,(1997) Meth. Enzymol. 276: 472-494) represented as spherically averagedelectron density (Table 1). Phases were improved and extended from 3.3 Åto 2.4 Å by solvent flipping (Abrahams et al. (1996) supra), and modelswere built from the data set.

C. Example 3 Preparation of Crystals of 50S Ribosomal Subunit/PuromycinComplex and Collection of X-Ray Diffraction Data

Crystals of 50S ribosomal subunits were grown and stabilized asdescribed earlier. CCdA-p-puromycin (see, FIG. 9A) was a generous giftfrom Michael Yarus (Welch et al. (1995) supra). Oligonucleotides fromamino-N-acylated minihelices (see, FIG. 9B) were synthesized byDharmacon. Following deprotection, the oligonucleotides were heatedbriefly to 100° C. and snap-cooled on ice to reanneal. Ribosomal 50Ssubunit crystals were stabilized and then soaked for 24 hours instabilization buffer plus 100 μM CCdA-p-puromycin or amino-N-acylatedmini-helices prior to cryovitrification in liquid propane and collectionof X-ray diffraction data. Phases were calculated by densitymodification (CNS) beginning with the best experimental phases using2F_(o)(analogue)−F_(o)(native) for amplitudes, from 60.0 to 3.2 Å.(Native amplitudes were from the most isomorphous native 1 data set,except for those amplitudes which were present only in the more completenative 2 data set. Calculated 2F_(o)−F_(o) amplitudes which were lessthan twice the corresponding calculated and were replaced byF_(o)(analogue)). Maps then were calculated using phases from densitymodified and 2F_(o)(analogue)−F_(o)(native) orF_(o)(analogue)−F_(o)(native) amplitudes.

D. Example 4 Antibiotic Binding Sites Located in the Polypeptide ExitTunnel Near the Peptidyl Transferase Center

Electron density maps derived from crystalline complexes of the H.Marismortui large subunit complexed with the three antibiotics tylosin,carbomycin A and anisomycin at about 3.0 Å resolution. All theseantibiotics bind to the ribosome in the region that lies between thepeptidyl transferase center as defined by the Yarus inhibitor,CCdA-p-puromycin, and the tips of the proteins L22 and L4 at the pointthat they form a small orifice in the polypeptide exit tunnel. Thegeneral location of this major antibiotic binding site is shown in FIG.19. Tylosin and carbomycin A appear to function by blocking the exit ofnewly synthesized polypeptides. Anisomycin appears to block the A-site.

The vast majority of the interactions between these antibiotics and theribosome are through rRNA that defines the A-site, and the surface ofthe tunnel between the peptidyl transferase center and protein L22.Since these antibiotics do not bind identically, there will be manyadditional ways that small molecule compounds can be designed to bind inthis region using the tools and methodologies of the present invention.For example, by connecting together components of each of the differentantibiotics which bind to non-overlapping sites it will be possible tocreate new hybrid antibiotics (see, Example 6). In addition, based onnew principles of small molecule RNA interaction shown by theseantibiotic complexes it is possible to design entirely novel smallmolecules that will bind to the same sites on the ribosome, as well asother potential RNA targets.

F. Example 5 Design and Testing of Hybrid Antibiotics

Many antibiotics that target ribosomes, more particularly largeribosomal subunits, and disrupt protein synthesis are complex moleculesthat are effectively concatenations of simpler substructures, at leastone of which interacts with a discrete part of the ribosome. When thecompound in question includes several interactive substructures, itsbinding site is effectively the sum of the subsites that contact andengage each such substructure. It has been found that many antibioticsthat target the large ribosomal subunit bind the ribosomal subunit atsites that are close to one another. Thus the possibility exists ofsynthesizing new antibiotics in which one ribosome-binding moiety of afirst known antibiotic is linked chemically to a ribosome-binding moietyof a second known antibiotic that interacts with an adjacent subsite.The new compound that results is thus a chimera or hybrid of the twoantibiotics from which it derives.

Chimeric antibiotics can be designed using the information about thestructures of antibiotic/ribosome complexes discussed hereinabove. Thesestructures permit the identification of antibiotic binding subsites inthe ribosome, and the specification of the chemical entities thatinteract with them. Equipped with such knowledge, those skilled in theart of organic synthesis can synthesize compounds that link thesubstructures of interest together in ways that should enable them tointeract with their respective subsites at the same time. Any compounddevised this way that functions in the manner intended is likely toinhibit cell growth and if it does, protein synthesis in vivo. At thevery least, it should block protein synthesis in in vitro assay systems.Further information about the ribosomal interactions of such a compoundcan be obtained by determining the structure of the complex it formswith the ribosome using the methods described in Section D, hereinabove.

For example, as a result of the work described herein, it has beendiscovered that the disaccharide moiety of carbomycin binds the largeribosomal subunit at a site in close proximity to the binding site for aportion of the anisomycin. Using this information and the softwarepackages described hereinabove, the skilled artisan can design a hybridantibiotic comprising the relevant ribosome binding portions ofcarbomycin and anisomycin linked by a suitable chemical linker.

FIG. 34 shows the design of another exemplary hybrid antibiotic. In thisfigure, a portion of the sparsomycin antibiotic is linked to a portionof the anisomycin antibiotic to produce a sparsoanisomycin hybridantibiotic. The portions of each antibiotic were chosen as a result ofthe structural studies herein which showed how each of the antibioticsbind the large ribosomal subunit. The hybrid antibiotic was designed topermit each component of the hybrid antibiotic to simultaneously bindits respective binding site in the large ribosimal subunit.

FIG. 35 shows the design of other exemplary hybrid antibiotics. In thisfigure, a portion of the sparsomycin antibiotic is linked to a portionof the chloromphenicol antibiotic to produce a sparsochloramphenicolhybrid antibiotic. In a first sparsochloramphenicol hybrid antibiotic(Hybrid A), n=1 in the linking region. In a second sparsochloramphenicolhybrid antibiotic (Hybrid B), n=2 in the linking region. The portion ofthe chloramphenicol molecule was chosen as a result of the structuralstudies described in Schlünzen et al. (2001) Nature 413: 814-821. Thehybrid antibiotic again was designed to permit each component of thehybrid antibiotic to bind its respective binding site in the largeribosomal subunit.

These hybrid molecules, once designed, can be synthesized and purifiedusing conventional synthetic organic chemistries and conventionalpurification schemes. Once synthesized and purified, the hybrid moleculeof interest can be screened for bioactivity and to determine, forexample, the IC₅₀ value for each molecule of interest. These screens caninclude, for example, growing micro-organisms on or in media eithersupplemented or lacking the hybrid molecule. Any reduction in the numberof micro-organisms or the size of colonies in the presence of the hybridmolecule would be indicative of bioactivity. Furthermore, the hybridmolecule could be tested in a cell-free coupledtranscription/translation assay or in translation system in the presenceof one or more labeled amino acids or using a nonradioactive reportersystem. Any reduction in the level of labeled amino acids incorporatedinto proteins in cell-free systems that include the hybrid moleculerelative to cell-free systems lacking the hybrid molecule would beindicative that the hybrid molecule acts as a functional proteinsynthesis inhibitor. It is contemplated that the hybrid molecule couldthen be iteratively refined as discussed hereinabove to enhance itsbioactivity and bioavailability.

E. Example 6 Binding of Macrolide Antibiotics to the Large RibosomalSubunit

Crystal structures of the H. Marismortui large ribosomal subunitcomplexed with five macrolides, carbomycin A, spiramycin, tylosin,azithromycin, and erythromycin show that these antibiotics bind in thepolypeptide exit tunnel immediately adjacent to the peptidyl transferasecenter. Tylosin, carbomycin A and spiramycin are 16-membered macrolides.Azithothromycin is a 15-membered macrolide. Erythromycin is a14-membered macrolide.

Their respective binding locations and bulk suggest that they inhibitprotein synthesis by blocking the passage of nascent polypeptidesthrough peptide exit tunnel. The saccharide at the C5 position of eachof the lactone rings extends up the exit tunnel towards the peptidyltransferase site. The isobutyrate extension of the carbomycin Adisaccharide overlaps the A-site substrate binding site. A reversiblecovalent bond appears to form between the ethylaldehyde substituent atthe C6 of the lactone ring and the N6 of A2103 (A2602, E. coli) in 23rRNA.

Crystals containing H. Marismortui large ribosomal subunits withcarbomycin A, spiramycin, tylosin, azithromycin, and erythromycin boundwere obtained adding stabilizing buffers containing these antibiotics topre-formed, large ribosomal subunit crystals. The structures of theseantibiotics complexed with these crystals were determined at 3.0 Åresolution (3.5 Å for erythromycin) from difference Fourier mapscalculated initially using observed diffraction amplitudes from thenative and complex crystals [F_(o)(complex)−F_(o)(native)], and then theco-ordinates of the entire complex were refined.

Ribosomes were purified and crystallized as described previously (Ban etal., (2000). Antibiotics were selected for testing based on their knownactivity against H. Marismortui (Sanz et al., (1993) Can. J Microbiol.39: 311-317) and their availability. Carbomycin A was obtained fromPfizer. Tylosin was purchased from Sigma. A mixture of spiramycins I, IIand II which differ only in the size of the moiety attached to C3, alsowere purchased from Sigma. Azithromycin was generously provided by DaleL. Boger, Department of Chemistry, the Scripps Research Institute, LaJolla, Calif. Erythromycin was obtained from Sigma.

Crystals containing H. Marismortui large ribosomal subunits complexedwith carbomycin A, spiramycin, tylosin, azithromycin and erythromycinwere obtained by soaking pre-formed, large-subunit crystals instabilizing buffers containing the antibiotics. Antibiotics weresolubilized in dimethylsulfoxide (DMSO), then added to the standardstabilization buffer (Ban et al., (2000) supra) to a final concentrationof 1.0 mM (and final DMSO of 1 to 4%), and then incubated at 4° C. for24 hours prior to cryo-vitrification of crystals in liquid propane.Initial x-ray diffraction data for the carbomycin A, spiramycin andtylosin antibiotics were collected at beamlines X25, X12b or X12c atBrookhaven National Laboratory. Data were reduced using denzo or HKL2000software and scaled with scalepack (Otwinowski (1997) “Processing ofX-ray Diffraction Data Collected In Oscillation Mode,” Methods inEnzymology 276(A):307-326). Electron density corresponding to thesemacrolides was first seen in F_(o)(antibiotic)−F_(o)(native) differenceFourier maps at 4.0 Å resolution. Higher resolution data were collectedat beamline ID19 at the Advanced Photon Source, Argonne NationalLaboratory. Beginning macrolide models and their topology and parameterfiles were constructed by connecting and modifying the lactone rings andsugars from various related small molecule structures (Woo et al. (1996)Tetrahedron 52(11): 3857-3872; Jones et al. (1982) Journal ofAntibiotics 35(4): 420-5; Stephenson et al. (1997) Pharmaceutical Sci.,86:1239) using standard stereochemical geometry and software xplo-2d(Kleywegt et al. (1998) Acta Cryst, D54: 1119-1131) and O (Jones et al.(1991) Acta Cryst. A47: 110-119). For the mixture of spiramycins, onlyspiramycin I was used in structure determination. The antibiotic modelsinitially were fit into F_(o)−F_(o) difference electron density maps.The structures of the antibiotics complexed with these crystals weredetermined at 3.0 Å resolution from difference Fourier maps calculatedinitially using observed diffraction amplitudes from the native andcomplex crystals, [F_(o)(complex)−F_(o)(native)], and then theco-ordinates of the entire complex were refined. The structures of thesecomplexes were refined using CNS (Brünger et al. (1998) Acta Cryst. D54:905-921) for rigid body refinement, energy minimization, and B-factorrefinement of the entire native ribosome structure including theantibiotic, and by manual modifications of only the immediate areasurrounding the bound antibiotic. Nonisomorphous differences distantfrom the antibiotic were ignored. The refinement process then wasrepeated iteratively on the antibiotic-containing model. The covalentstructures of the macrolides and a conformational change involving A2103accounted for the principal features in the difference electron densitymaps.

Based on these studies, the ethylaldehyde group at the C6 position ofthe lactone ring of each of the 16-membered macrolides appears to form acovalent bond with the N6 of A2103. Not only are the N6 of A2103 and theethylaldehyde at C6 of the macrolides juxtaposed, they are joined bycontinuous electron density, which, at 3.0 Å resolution, is indicativeof chemical bonding. In contrast, for both the 15-membered and14-membered macrolides (both of which lack aldehyde groups) no suchcontinuous electron density was observed. The only models for themacrolide-ribosome complex that fit the observed electron densitysatisfactorily are those in which the N6 of A2103 is covalently bondedto the carbon of the aldehyde group of the macrolide. Aldehydes reactreversibly with primary amines, and if the resulting carbinolaminesdehydrate, Schiff's bases are produced. However, when the exocyclicamine of a nucleotide base is involved, the expected product is acarbinolamine, not a Schiff's base (McGhee et al. (1977) Biochem. 14(6):1281-1303). Therefore, the model that best fits the out of planeelectron density in this region links them via a carbinolamine, not aSchiff's base.

Based upon these studies, it has been found that carbomycin A,spiramycin, tylosin, azithromycin, and erthyromycin all bind in thepeptide exit tunnel of the large ribosomal subunit, immediately adjacentto the peptidyly transferase center. The five co-crystal structures weresuperimposed based only on the phosphates of the ribosomal RNA in orderto objectively compare their relative binding. The three 16-memberedmacrolides superimpose on almost an atom by atom basis. Furthermore, allfive lactone rings occupy a very similar position, share a commonconformation and orientation, and form similar interactions with theribosome. In addition, shared moieties such as the mycaminose of the16-membered rings (and the corresponding desosamine of the othermacrolides) superimpose on almost an atom by atom basis. All the sugearmoieties assume the same relaxed extended conformations with respect tothe lactone ring that are observed in small molecule macrolidestructures.

Based on the resolved structures, it appears that hydrophobicinteractions are important in macrolide binding. One face of the lactonerings of these antibiotics is quite hydrophobic, while the opposite faceis more hydrophilic in character. All five of these macrolides bind tothe ribosome with the hydrophobic faces of their lactone rings facingthe wall of the exit tunnel and the hydroplilic faces of their lactonerings exposed to solution. The tunnel wall binding site includes thearomatic face of G2646, which is exposed because the C2098-G2646 basepair is helix terminating.

In addition, these structures shed light on why the lengths of theoligopeptides synthesized by macrolide-poisoned ribosomes vary the waythey do. The length of the oligopeptides synthesized in the presence ofmacrolide inhibitors is determined by the extent to which thesubstituents at the C5 position of the lactone ring penetrate thepeptidyl transferase center. Erythromycin and azithromycin, which haveonly a monosaccharide at this position, should permit the synthesis oflonger peptides than tylosin or spiramycin, which have a disaccharide.These observations are consistent with those derived from biochemicalstudies which indicate that erythromycin does permit the formation oftetrapeptides, tylosin and spiramycin allow formation of onlydipeptides, and carbomycin A strongly inhibits the formation of even thefirst peptide bond.

INCORPORATION BY REFERENCE

The disclosure of each of the patent documents, scientific articles,atomic-co-ordinates (including, without limitation, those sets depositedat the Research Collaboratory for Structural Bioinformatics Protein DataBank (PDB) with the accession numbers PDB ID: 1FFK; PDB ID: 1FFZ; PDBID: 1FG0; PDB ID: 1JJ2; PDB ID: 1K73; PDB ID: 1KC8; PDB ID: 1K8A; PDBID: 1KD1; and PDB ID: 1K9M, and/or contained on Disk No. 1) referred toherein is incorporated by reference herein.

All materials submitted herewith on compact disk, Disk No. 1 areincorporated by reference herein. Disk No. 1 was created on Feb. 6, 2002and is identified as containing the following thirty-nine files:

File: Size(in bytes) <DIR> <DIR> 1JJ2.RTF 12,742,023 1JJ2.TXT 8,372,780ANISOMYCIN.PDB 7,593,128 azithromycin.pdb 8,088,411 BLASTICIDIN.PDB7,594,206 CARBOMYCIN.PDB 7,592,552 crythromycin.pdb 7,690,337 FOLDERA<DIR> FOLDERB <DIR> FOLDERC <DIR> linezolid.pdb 8,086,197 PDB1FFK.DOC7,046,656 PDB1FFK.ENT 5,484,652 PDB1FFZ.DOC 1,219,072 PDB1FFZ.ENT937,342 PDB1FG0.DOC 1,225,728 PDB1FG0.ENT 942,344 SPARSOMYCIN.PDB7,541,230 SPIRAMYCIN.PDB 7,592,549 TYLOSIN.PDB 7,595,512VIRGINIAMYCIN.PDB 7,591,745 FOLDERA: <DIR> <DIR> 1JJ2.PDB 8,270,586FOLDERB: <DIR> <DIR> ANISOMYC.PDB 8,383,598 BLASTICI.PDB 8,393,766CARBOMYC.PDB 8,387,042 SPARSOMY.PDB 7,593,360 SPIRAMYC.PDB 8,402,048TYLOSIN.PDB 8,339,400 VIRGINIA.PDB 7,590,514 FOLDERC: <DIR> <DIR>AZITHROM.PDB 7,989,198 LINEZOLI.PDB 7,987,583

EQUIVALENTS

The invention may be embodied in other specific forms without departingform the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A method of identifying a molecule that binds to a large ribosomalsubunit, the method comprising the steps of: (a) providing a molecularmodel comprising one or more target regions selected from the groupconsisting of at least a portion of (i) a peptidyl transferase site,(ii) an A-site, (iii) a P-site, (iv) an E-site, (v) an elongation factorbinding domain, (vi) a polypeptide exit tunnel, and (vii) a signalrecognition particle (SRP) binding domain, from the atomic co-ordinatesfor Haloarcula marismortui large ribosomal subunit found on Disk 1underfile name 1JJ2.RTF, 1JJ2.TXT, 1JJ2.PDB, PDB1FFK.DOC, or PDB1FFK.ENT, ordeposited at the Protein Data Bank under accession number PDB ID: 1JJ2or 1FFK, or derived from said Haloarcula marismortui atomic co-ordinatesby molecular modeling; (b) using the molecular model to identify acandidate molecule that can bind to the one or more target regions inthe molecular model; and (c) producing the candidate molecule identifiedin step (b).
 2. The method of claim 1, comprising the additional step ofdetermining whether the candidate molecule modulates ribosomal activity.3. The method of claim 2, comprising the additional step of repeatingsteps (a) through (c) to identify and produce a modified candidatemolecule having higher binding specificity, higher binding affinity orhigher potency relative to the candidate molecule.
 4. The method ofclaim 3, comprising the additional step of determining whether themodified candidate molecule modulates ribosomal activity.
 5. The methodof claim 4, comprising the additional step of, after determining whetherthe modified candidates molecule modulates ribosomal activity, producinga large quantity of the modified candidates molecule that modulatesribosomal activity.
 6. The method of claim 1, wherein the one or moretarget regions comprises at least a portion of a peptidyl transferasesite.
 7. The method of claim 6, wherein the peptidyl transferase site isdefined by a plurality of residues set forth in Table 5A or Table 5B. 8.The method of claim 1, wherein the one or more target regions comprisesat least a portion of an A-site.
 9. The method of claim 8, wherein theA-site is defined by a plurality of residues set forth in Table 6A orTable 6B.
 10. The method of claim 8, wherein the one or more targetregions further comprises at least a portion of a P-site.
 11. The methodof claim 10, wherein the one or more target regions further comprises atleast a portion of a polypeptide exit tunnel.
 12. The method of claim 8,wherein the one or more target regions further comprises at least aportion of a polypeptide exit tunnel.
 13. The method of claim 1, whereinthe one or more target regions comprises at least a portion of a P-site.14. The method of claim 13, wherein the P-site is defined by a pluralityof residues set forth in Table 7A or Table 7B.
 15. The method of claim1, wherein the one or more target regions comprises at least a portionof a polypeptide exit tunnel.
 16. The method of claim 15, wherein theexit tunnel is defined by a plurality of residues set forth in Table 8A,Table 8B, Table 9, or Table
 10. 17. The method of claim 1, wherein themolecular model is in an electronic form.
 18. The method of claim 1,wherein the molecular model is generated from atomic co-ordinatesproduced by molecular modeling.
 19. The method of claim 1 or 18, whereinthe molecular model is generated from atomic co-ordinates produced byhomology modeling using at least a portion of the atomic co-ordinatesdeposited at the Protein Data Bank under accession number PDB ID: 1FFK,1FFZ, 1FG0, 1JJ2, or recorded on Disk No. 1under file name PDB1FFK.DOC,PDB1FFK.ENT, PDB1FFZ.DOC, PDB1FFZ.ENT, PDB1FG0.DOC, PDB1FG0.ENT,1JJ2.RTF, 1JJ2.TXT, or 1JJ2.PDB.
 20. The method of claim 1 or 18,wherein the molecular model is generated from atomic co-ordinatesproduced by molecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, 1JJ2, or Disk No. 1under file namePDB1FFK.DOC, PDB1FFK.ENT, PDB1FFZ.DOC, PDB1FFZ.ENT, PDB1FG0.DOC,PDB1FG0.ENT, 1JJ2.RTF, 1JJ2.TXT, or 1JJ2.PDB.
 21. The method of claim 1,wherein the molecular model comprises residues that are conserved amongone or more prokaryotic organisms.
 22. The method of claim 1, whereinthe molecular model comprises a residue that is present in a prokaryoticribosome but is absent from a eukaryotic ribosome or a eukaryoticmitochondrial ribosome.
 23. The method of claim 22, wherein theeukaryotic ribosome is a mammalian ribosome.
 24. A method of identifyinga molpcule that binds to a large ribosomal subunit, the methodcomprising the steps of: (a) providing a molecular model comprising oneor more target regions selected from the group consisting of a peptidyltransferase site, an A-site, a P-site, an E-site, an elongation factorbinding domain, a polypeptide exit tunnel, and a signal recognitionparticle (SRP) binding domain, from the atomic co-ordinates (i) forHaloarcula marismortui large ribosomal subunit found on Disk 1 underfile name 1JJ2.RTF, 1JJ2.TXT, 1JJ2.PDB, PDB1FFK.DOC, or PDB1FFK.DOC, ordeposited at the Protein Data Bank under accession number PDB ID: 1JJ2or 1FFK, or (ii) derived from the Haloarcula marismortui atomicco-ordinates by molecular modeling; (b) using the molecular model toidentify a candidate molecule that can bind to the one or more targetregions in the molecular model; and (c) producing the candidate moleculeidentified in step (b).